# Patent

## Title
Pro-angiogenic genes in ovarian tumor endothelial cell isolates

## Abstract

A gene profiling signature for ovarian tumor endothelial cells is disclosed herein. The gene signature can be used to diagnosis or prognosis an ovarian tumor, identify agents to treat an ovarian tumor, to predict the metastatic potential of an ovarian tumor and to determine the effectiveness of ovarian tumor treatments. Thus, methods are provided for identifying agents that can be used to treat ovarian cancer, for determining the effectiveness of an ovarian tumor treatment, or to diagnose or prognose an ovarian tumor. Methods of treatment are also disclosed which include administering a composition that includes a specific binding agent that specifically binds to one of the disclosed ovarian endothelial cell tumor-associated molecules and inhibits ovarian tumor in the subject.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This is a continuation-in-part application of International Patent Application PCT/US2008/054014, filed Feb. 14, 2008, designating the United States and published in English as WO 2008/101118, which claims the benefit of U.S. Provisional Application No. 60/901,455, filed on Feb. 14, 2007. The entire contents of these prior applications are incorporated herein by reference.


ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under contract CA083639 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE DISCLOSURE
This disclosure relates to the field of ovarian cancer and in particular, to methods for treating ovarian cancer by targeting ovarian endothelial cell tumor-associated molecules identified by an ovarian tumor endothelial cell gene expression profile and methods for identifying therapeutic agents.
BACKGROUND
Ovarian cancer is the fifth most common form of cancer in women in the United States, accounting for three percent of the total number of cancer cases and twenty-six percent of those occurring in the female genital tract. The American Cancer Society estimated that 15,310 deaths would be caused in women living in the United States in 2006. A large majority of women who die of ovarian cancer will have had serous carcinoma of the ovarian epithelium, a condition which occurs in sixty percent of all cases of ovarian cancer (Boring et al., Cancer J. Clin. 44: 7-26, 1994).
Women with ovarian cancer are typically asymptomatic until the cancer has metastasized. As a result, most women with ovarian cancer are not diagnosed until the cancer has progressed to an advanced and usually incurable stage (Boente et al., Curr. Probl. Cancer 20: 83-137, 1996). Survival rates are much better in women diagnosed with early-stage ovarian cancers, about ninety percent of these women are still alive five years after diagnosis.
Treatment of ovarian cancer typically involves a variety of treatment modalities. Generally, surgical intervention serves as the basis for treatment (Dennis S Chi & William J. Hoskins, Primary Surgical Management of Advanced Epithelial Ovarian Cancer, in Ovarian Cancer 241, Stephen C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). Treatment of serous carcinoma often involves cytoreductive surgery (hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and lymphadenectomy) followed by adjuvant chemotherapy with paclitaxel and either cisplatin or carboplatin (Eltabbakh, G. H. & Awtrey, C. S., Expert Op. Pharmacother. 2(10): 109-24, 2001).
Despite a clinical response rate of 80% to primary treatment with surgery and chemotherapy, most subjects experience tumor recurrence within two years of treatment. The overwhelming majority of subjects will eventually develop chemoresistance and die as a result of their cancer. Thus, a need exists to identify alternative treatments for ovarian cancer.
SUMMARY OF THE DISCLOSURE
A gene profiling signature is disclosed herein that can be used to predict clinical outcome and develop therapeutics for treating ovarian cancer in a subject. For example, the ovarian endothelial cell tumor-associated molecules identified by the gene profile signature can serve as prognostic indicators as well as targets for specific therapeutic molecules that can reduce or eliminate ovarian cancer. Thus, methods of identifying an agent for treating an ovarian tumor are provided. In some examples, the methods include contacting a cell, such as an ovarian tumor cell or an ovarian tumor endothelial cell, with one or more test agents under conditions sufficient for the one or more test agents to alter the activity of at least one ovarian endothelial cell tumor-associated molecule listed in any of Tables 1-5. The method includes detecting the activity of the at least one ovarian endothelial cell tumor-associated molecule in the presence and absence of the one or more test agents. The activity of the at least one ovarian endothelial cell tumor-associated molecule in the presence of the one or more test agents is then compared to the activity in the absence of such agents to determine if there is differential expression of the at least one ovarian endothelial cell tumor associated molecule. Differential expression of the ovarian endothelial cell tumor-associated molecule in the presence of the test agent(s) indicates that the one or more test agents can be used to treat an ovarian tumor.
Methods are also provided for treating an ovarian tumor. In some examples, the method includes administering to the subject a therapeutically effective treatment to inhibit ovarian tumor growth. In an example, the treatment includes administering a therapeutically effective amount of a specific binding agent that binds with high specificity to one of the ovarian endothelial cell tumor-associated molecules listed in Tables 1, 2, 4 or 5 and alters expression or activity of the molecules, thereby treating the ovarian tumor in the subject (for example, by decreasing tumor vascular growth, tumor growth or tumor volume). In particular examples, the specific binding agent preferentially binds to and inhibits expression or activity of one of the ovarian endothelial cell tumor-associated molecules that is upregulated in an ovarian tumor endothelial cell, such as Zeste homologue 2 (EZH2), EGF-like domain, multiple 6 (EGFL6), tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), Twist homologue 1 (TWIST1), stanniocalcin 1 (STC1), homeodomain-only protein (HOP), chondroitin sulfate proteoglycan 2 (CSPG2), and plexin domain containing 1 (PLXDC1). In other particular examples, ovarian tumor growth is inhibited by the specific binding agent preferentially binding to and inhibiting expression of one of the ovarian endothelial cell tumor-associated molecules listed in any of Tables 1, 2, 4 or 5 which are involved in angiogenesis, such as molecules involved in cell proliferation, tube formation or cell motility and are upregulated in ovarian tumor endothelial cells.
Methods are also provided for determining the effectiveness of an agent for the treatment of an ovarian tumor in a subject with the ovarian tumor. In an example, the method includes detecting expression of an ovarian endothelial cell tumor-associated molecule in a sample from the subject following administration of the agent. The expression of the ovarian endothelial cell tumor-associated molecule following administration is compared to a control, such as specific binding agents that bind to and inhibit one of the ovarian endothelial cell tumor-associated molecules listed in any of Tables 1, 2, 4 or 5 that is upregulated in ovarian endothelial tumor cells. An alteration in the expression of the ovarian endothelial cell tumor-associated molecule (such as a decrease in expression of a molecule upregulated in ovarian tumor endothelial cells or an increase in expression of a molecule downregulated in such cells) following treatment indicates that the agent is effective for the treatment of the ovarian cancer in the subject. In a specific example, the method includes detecting and comparing the protein expression levels of the ovarian endothelial cell tumor-associated molecules. In other examples, the method includes detecting and comparing the mRNA expression levels of the ovarian endothelial cell tumor-associated molecules.
Methods of diagnosing and prognosing an ovarian tumor (such as a tumor that overexpresses at least one of the disclosed ovarian endothelial cell tumor-associated molecules) are provided. In some examples, such methods are performed prior to the treatment methods described herein. However, such methods can also be used independently of the disclosed treatment methods. In particular examples, the method includes determining the metastatic potential of an ovarian tumor in a subject by detecting expression of at least one ovarian endothelial cell tumor-associated molecule in a sample obtained from a subject with an ovarian tumor. The at least one ovarian endothelial cell tumor-associated molecule is involved in promoting angiogenesis, such as cell proliferation, cell motility or tube formation, such as EZH2. The method further includes comparing expression of the at least one ovarian endothelial cell tumor-associated molecule in the sample obtained from the subject with the ovarian tumor to a control. An alteration in the expression of the at least one ovarian endothelial cell tumor-associated molecule involved in promoting angiogenesis indicates that the subject has an ovarian tumor with increased metastatic potential.
In additional examples, methods are disclosed for predicting a clinical outcome in a subject with an ovarian tumor, such as advanced stage epithelial ovarian cancer. In an example, the methods include detecting expression of at least one ovarian endothelial cell tumor-associated molecules listed in Tables 1-5 or combinations thereof (such as at least 1, at least 3, at least 5 or at least 10 of such molecules) in a sample obtained from the subject with the ovarian tumor. The methods also can include comparing expression of the at least one ovarian endothelial cell tumor-associated molecule in the sample obtained from the subject with the ovarian tumor to a control (such as a normal sample or value representing such expression expected in a normal sample), wherein an alteration in the expression of the at least one ovarian endothelial cell tumor-associated molecule indicates that the subject has a decreased chance of survival. For example, an alteration in the expression, such as an increase in the expression of EZH2 indicates a poor prognosis, such as a decreased chance of survival. In one example, a decreased chance of survival includes a survival time of equal to or less than a year. Alterations in the expression can be measured using methods known in the art, and this disclosure is not limited to particular methods. For example, expression can be measured at the nucleic acid level (such as by real time quantitative polymerase chain reaction or microarray analysis) or at the protein level (such as by Western blot analysis).
The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a graph illustrating the comparative fold change in relative expression levels between microarray data and real-time quantitative RT-PCR data of selected genes from the pro-angiogenic gene signature provided in Table 1.
FIG. 2 is a graph illustrating protein expression in ovarian endothelial cells for a subset of the proteins detected following staining of samples with immunofluorescently-labeled PTK2, Fyn, MMP-9, β2-arrestin, Jagged1 and PLXDC1.
FIGS. 3A, 3B and 3C are digital images of siRNA-mediated silencing of (FIG. 3A) EZH2, (FIG. 3B) Jagged1 and (FIG. 3C) protein tyrosine kinase 2 (PTK2), as assessed using Western blot analyses.
FIG. 3D is a graph illustrating the effect of EZH2, Jagged1 or PTK2 silencing on human umbilical vein endothelial cell (HUVEC) tube formation.
FIG. 3E is a graph illustrating the effect of EZH2 silencing on HUVEC migration.
FIG. 3F is a graph illustrating the effect of Jagged1 silencing on HUVEC migration.
FIG. 3G is a graph illustrating the effect of PTK2 silencing on HUVEC migration.
FIG. 4 is a digital image illustrating down regulation of EZH2 by mouse EZH2 siRNA in mouse ovarian endothelial cells.
FIGS. 5A, 5B and 6 provide graphs illustrating the therapeutic effects of siRNA-mediated EZH2 down regulation on HeyA8 (FIG. 5A) and SKOV3ip1 (FIGS. 5B and 6) ovarian tumors.
FIGS. 7A-7H illustrate EZH2 expression in human ovarian carcinoma. FIG. 7A provides digital images representative of human tumors with low and high EZH2 expression based on immunohistochemical staining. FIG. 7B provides Kaplan-Meier curves of disease-specific mortality for patients whose ovarian tumors expressed high and low levels of EZH2 (EZH2-T). The log-rank test (two-sided) was used to compare differences between the two groups. Increased EZH2-T was significantly associated with decreased overall survival (p<0.001). FIG. 7C provides digital images representative of human ovarian tumor vasculature (arrowheads point to endothelial cells) with low and high immunohistochemical staining for EZH2. FIG. 7D provides Kaplan-Meier curves of disease-specific mortality of patients whose ovarian vasculature expressed low versus high EZH2 (EZH2-Endo) EZH2-Endo was predictive of poor overall survival. FIG. 7E provides digital images representative of human ovarian tumors with low or high immunohistochemical staining for VEGF. FIG. 7F provides a bar graph VEGF expression was strongly associated with high EZH2-Endo (*p<0.01). FIG. 7G provides digital images representative of human ovarian tumors with low or high immunohistochemical staining for microvessel density (MVD). FIG. 7H provides a bar graph illustrating high MVD counts in a tumor were significantly associated with high EZH2-Endo expression (*p<0.001). Images in panels A, C, and E were taken at original magnification ×200, and in panel g at original magnification ×200.
FIGS. 8A-8C are graphs illustrating VEGF-increased EZH2 expression in endothelial cells. Results in FIGS. 8A and 8B are in response to 6-hour treatments with EGF (25 ng/μL), VEGF (50 ng/μL), conditioned medium (CM) from the non-cancerous ovarian epithelial cell line IOSE120, two ovarian cancer cell lines OVCA420 and SKOV3, and complete medium with either 10% serum (A) or 2% serum (B). Percent fold changes represent the mean+/−s.d. of triplicate experiments compared to untreated control cells. *p<0.05; **p<0.01; ***p<0.001. FIG. 8A illustrates that EZH2 promoter activity is increased in an endothelial cell line in response to EGF, VEGF, and conditioned media from ovarian cancer cell lines. EAhy926 hybridoma endothelial cell line was cotransfected with the Renilla luciferase plasmid and firefly luciferase plasmid either with or without the EZH2 promoter construct followed by treatment with EGF, VEGF and conditioned medium and promoter activity was determined. FIG. 8B illustrates that EZH2 mRNA levels are increased in HUVEC in response to EGF, VEGF, and conditioned media from ovarian cancer cell lines. Cells were treated as indicated and purified RNA was used in real-time quantitative RT-PCR. Control values were normalized using 3 housekeeping genes. FIG. 8C Pearson's analysis shows significant correlation between EZH2 and VEGF expression values (Log2) from 29 microdissected high-grade serous papillary ovarian adenocarcinomas.
FIGS. 9A-9E show EZH2 gene silencing increases VASH1 mRNA expression in endothelial cells. FIG. 9A is a digital image of a polyacrylamide gel illustrating PCR products generated by as ChIP assay of EZH2 binding to human VASH1 promoter in response to VEGF in HUVEC. Cross-linked chromatin from HUVEC was treated with (+) or without (−) VEGF and immunoprecipitated (IP) using EZH2 or mouse IgG antibodies. The input and immunoprecipitated DNA were subjected to PCR using primers corresponding to the 3800 to 3584 base pairs upstream of VASH1 transcription start site. PCR products were examined on ethidium bromide-stained agarose gel. FIG. 9B is a bar graph illustrating EZH2 mRNA levels in cells transfected with control or mouse EZH2 siRNA and harvested after 72 hours. RNA was isolated and subjected to real-time quantitative RT-PCR. The fold difference in levels of EZH2 mRNA represents the mean of triplicate experiments compared to control siRNA treated cells. Error bars represent s.e.m. *p<0.05. FIG. 9C is a bar graph illustrating the fold difference in levels of VASH1 mRNA as compared to control siRNA treated cells. Error bars represent s.e.m. *p<0.01. FIG. 9D illustrates the effect of EZH2 gene silencing on methylation status of VASH1 in VEGF-treated MOECs as detected by methylation specific PCR. The inhibitory units of methylated VASH1 were normalized by that of the un-methylated VASH1 and represent the mean of triplicate experiments. FIG. 9E is a digital image of a Western blot of lysate collected 48 hours after transfection of MOEC with control, VEGF treated and mouse EZH2 siRNA treated cells.
FIGS. 10A-10C show E2F transcription factors increases upon VEGF treatment in MOEC. FIG. 10A is a bar graph illustrating expression levels of E2F transcription factors in MOEC. Cells were treated with VEGF for 6 hours and subjected to Q-RT-PCR. FIG. 10B provides a pair of bar graphs illustrating silencing of E2F1, E2F3 and E2F5 transcription factors by targeted siRNA in MOEC. Cells were transfected with corresponding siRNAs. After 24 hours and 48 hours, cells were collected; RNA was isolated and was subjected to real-time Q-RT-PCR. E2F3 and E2F5 gene silencing decreases EZH2 expression levels. EZH2 expression levels were analyzed in E2F1, E2F3 and E2F5 silenced samples using Q-RT-PCR. The fold difference in levels of mRNA expression represents the mean of triplicate experiments compared to cells (A) and VEGF treated cells (B). Error bars represent s.e.m. *p<0.01. FIG. 10C is a bar graph illustrating the effect of VASH1 gene silencing on tube formation in endothelial cells. HUVECs were plated on Matrigel after transfecting the cells with either control or human VASH1 siRNA. Vascular tube formation was evaluated by microscopic observation.
FIGS. 11A-11E illustrate the physical characteristics of siRNA/CH nanoparticles. FIG. 11A is a table providing the composition of CH/TPP/siRNAs. FIG. 11B is a graph illustrating the mean particle size of siRNA/CH particles as measured using light scattering with a particle analyzer, showing that nanoparticles maintained 100-200 nm size up to 7:1 ratio (CH:TPP). FIG. 11C is a graph illustrating that zeta potential of siRNA/CH nanoparticles showed slight positive charge. FIG. 11D is a graph illustrating incorporation efficiency of siRNA into CH nanoparticles with 3:1 ratio of CH:TPP resulting in >75% incorporation efficiency. FIG. 11E is a digital image following atomic force microscopy (AFM) demonstrating that siRNA/CH nanoparticles were spherical and <150 nm in size.
FIGS. 12A-12E illustrate incorporation, stability and intracellular uptake of siRNA/CH nanoparticles. FIG. 12A is a digital image illustrating electrophoretic migration of naked siRNA and siRNA/CH nanoparticles. SiRNA/CH nanoparticles (open arrow) remained at top of the gel compared to naked siRNA (solid arrow), which migrated downward. FIG. 12B is a digital image illustrating electrophoretic migration of siRNA/CH nanoparticles in the presence of 50% serum. SiRNA/CH nanoparticles were collected at different time points of incubation at 37° C. (Lane 1; naked siRNA, Lanes 2 to 5; siRNA/CH nanoparticles). Naked siRNA (solid arrow) was degraded over 12 to 24 hours in serum containing media; whereas CH nanoparticles (open arrow) protected the siRNA from degradation in serum. Increased binding efficiency of siRNA/CH nanoparticles was noted compared to naked siRNA. FIG. 12C is a fluorescence microscopy digital image of HeyA8 cells after incubating either with siRNA alone or with siRNA/CH nanoparticles at 4° C. for 20 minutes in PBS. FIG. 12D is a series of tracings from a flow cytometry analysis demonstrating that uptake efficiency of nanoparticles into cells was increased by 72-fold after incubating cells in PBS at 4° C. for 20 minutes. FIG. 12E is graphical representation of percentage of uptake of Alexa-555 siRNA by cells by flow cytometry analysis.
FIGS. 13A-13E illustrate in vivo siRNA delivery using CH nanoparticles and the distribution of siRNA following single intravenous injection of Alexa-555 siRNA/CH nanoparticles in orthotopic HeyA8 tumor bearing nude mice. FIG. 13A provides a pair of digital images illustrating fluorescent siRNA distribution in tumor tissue of hematoxylin and eosin, original magnification ×200 (left); stained with anti-CD31 (green) antibody to detect endothelial cells (right). FIG. 13B provides a pair of digital images of 50-μm sections stained with Cytox Green and examined with confocal microscopy (original magnification ×400) (left); lateral view (right) Images taken every 1 μm were stacked and examined from the lateral view. Nuclei were labeled green and fluorescent siRNA (red) was seen throughout the section. At all time points, punctated emissions of the siRNA were noted in the perinuclear regions of individual cells, and siRNA was seen in >80% of fields examined. (c) Western blot of lysates from orthotopic tumors collected 24, 48, 72 and 96 hours after a single injection of control siRNA/CH or human (EZH2 Hs siRNA/CH). FIG. 13D provides multiple digital images illustrating EZH2 gene silencing in HeyA8 tumor as well as tumor endothelial cells. Tumors were collected after 48 hours of single injection of control siRNA/CH, or EZH2 Hs siRNA/CH, or EZH2 Mm siRNA/CH and stained for EZH2 (green) and CD31 (red). Images were taken at original magnification, ×200. FIG. 13E is a pair of graphs illustrating the effects of EZH2 Hs siRNA/CH or EZH2 Mm siRNA/CH on tumor weight in mouse orthotopic tumor models. Nude mice were injected with HeyA8 or SKOV3ip1 ovarian cancer cells and 1 week later, were randomly assigned (10 mice per group) to receive therapy: (1) control siRNA/CH, (2) EZH2 Hs siRNA/CH, (3) EZH2 Mm siRNA/CH, and (4) combination of EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH. Mice were sacrificed when any animals in control or a treatment group became moribund (after 3 to 4 weeks of therapy) and mouse weight, tumor weight and tumor location were recorded. Error bars represent s.e.m. *p<0.05; **p<0.001.
FIG. 14 provides a series of digital images of Western blots of lysate collected 72 hours after transfection of HeyA8 cells or MOEC with control, human EZH2, or mouse EZH2 siRNA.
FIG. 15 is a pair of graphs illustrating the weight distribution of HeyA8 and SKOV3ip1 tumors. Seven days following tumor cell injection, mice were randomly divided into 4 groups (10 mice per group) to receive therapy: (1) control siRNA/CH, (2) EZH2 Hs siRNA/CH, (3) EZH2 Mm siRNA/CH, and (4) combination of EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH. Mice were sacrificed when any animals in control or a treatment group became moribund (after 3 to 4 weeks of therapy) and tumor weight was recorded.
FIGS. 16A-16B (A) Effect of tumor (EZH2 Hs siRNA/CH) or endothelial (EZH2 Mm siRNA/CH) targeted EZH2 siRNA on MVD and pericyte coverage. Tumors harvested following 3 to 4 weeks of therapy were stained for CD31 (MVD; red) and desmin (pericyte coverage; green). All pictures were taken at original magnification ×200. The bars in the graphs correspond sequentially to the labeled columns of images at left. Error bars represent s.e.m. *p<0.05; **p<0.001. (B) Effects of VASH1 gene silencing on tumor growth in vivo. Nude mice were injected with SKOV3ip1 ovarian cancer cells and 1 week later, were randomly divided into 5 groups (10 mice per group): (1) control siRNA/CH, (2) EZH2 Mm siRNA1/CH, (3) EZH2 Mm siRNA2/CH (4) EZH2 Mm siRNA3/CH (5) VASH1 Mm siRNA1/CH and (6) combination of EZH2 Mm siRNA1/CH plus VASH1 Mm siRNA/CH.
FIG. 16B is a bar graph illustrating the number of cells that migrated in the presence and absence of VASH1 siRNA. Mice were sacrificed when any animals in control or a treatment group became moribund (after 3 to 4 weeks of therapy) and mouse weight, tumor weight and tumor location were recorded. Error bars represent s.e.m. *p<0.05.
FIG. 17 illustrates the effects of EZH2 Hs siRNA/CH or EZH2 Mm siRNA/CH on proliferation. Tumors were harvested following 3-4 weeks of therapy and then stained for proliferating cell nuclear antigen (PCNA). All images were taken at original magnification ×100. The bars in the graphs correspond sequentially to the labeled columns of images at left. Error bars represent s.e.m. *p<0.05.



SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NOS: 1, 6-27, and 44-47 are nucleic acid sequences of exemplary primers.
SEQ ID NOS: 2-5 and 28-43 are nucleic acid sequences of exemplary siRNAs.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Despite improvements in surgery and chemotherapy, mortality rates in women with advanced ovarian carcinoma have remained largely unchanged (Cannistra, N. Engl. J. Med. 329: 1550-1559, 1993). Therefore, novel therapeutic strategies are needed. Growth of tumors, both at the primary and metastatic sites, requires a blood supply for expansion beyond 1-2 mm (Folkman, J. Nat. Canc. Inst. 82: 4-6, 1990). Targeting tumor angiogenesis by inhibiting endothelial cells that support tumor growth is particularly promising because of their presumed genetic stability. The recent success of a humanized monoclonal antibody bevacizumab (trade name Avastin®) against vascular endothelial growth factor in prolonging the lives of patients with advanced colon and breast carcinoma demonstrates the promise of such approaches (Hurwitz et al., N. Engl. J. Med. 350: 2335-2342, 2004 and Jain et al., Nat. Clin. Pract. Oncol. 3: 24-40, 2006). However, the full spectrum of differences in the tumor vasculature compared to its normal counterpart is not known. Identification of additional targets on tumor endothelium may allow opportunities for developing new therapeutic approaches to inhibit angiogenesis in a tumor-specific manner.
In recent years, whole genome expression profiling of cancer using methods such as microarray and serial analysis of gene expression (SAGE) have provided insight into the molecular pathways involved in cancer onset and progression. While selected genes in ovarian cancer vasculature have been characterized, there is little information regarding global gene expression alterations in ovarian cancer endothelium.
Disclosed herein is a gene expression signature identifying endothelial cell tumor-associated molecules in ovarian tumor endothelial cell isolates. Endothelial cells were purified from human ovarian tissues and invasive ovarian epithelial cancers, and a gene expression profile was established for ovarian tumor endothelial cells using microarray analyses. The gene expression profile disclosed herein identifies genes whose expression is differentially regulated in tumor versus normal endothelial cells. This profile reveals distinct expression profiles for tumor endothelial cell isolates as compared to non-tumor endothelial isolates.
The disclosed gene expression profile also reveals genes and collections or sets of genes that serve as effective molecular markers for angiogenesis in ovarian cancer, predict clinical outcome as well as such genes or gene sets that can provide clinically effective therapeutic targets for ovarian cancer. This has significant implications for the treatment of ovarian cancer. For example, methods are disclosed for treating ovarian cancer (for example, reducing or inhibiting ovarian cancer growth by targeting ovarian endothelial cell tumor-associated molecules, such as molecules believed to be involved in angiogenesis). For example, molecules involved in cell motility, tube formation or cell proliferation can be identified by the gene profile signature. In an example, a therapeutically effective amount of a specific binding agent is administered to a subject. For example, the specific binding agent preferentially binds to one or more of the identified ovarian endothelial cell tumor-associated molecules listed in any of Tables 1, 2, 4, 5 or a combination thereof to alter the expression or activity of such molecule (e.g., increase expression or activity of a molecule that is downregulated in ovarian endothelial tumor cells or decrease expression or activity of a molecule that is upregulated in such cells). In one example, the specific binding agent preferentially binds to one or more of the identified ovarian endothelial cell tumor-associated molecules listed in any of Tables 1, 2, 4, 5 or a combination thereof that are upregulated in ovarian endothelial tumor cells (as indicated by a positive fold change in Table 1) to decrease expression or activity of the one or more molecules. As a result, ovarian cancer in the subject is thereby reduced or eliminated. In a particular example, the specific binding agent is an inhibitor, such as a siRNA, of one or more of the disclosed ovarian endothelial cell tumor-associated molecules described in any of Table 1, 2, 4 or 5 whose expression is upregulated in ovarian endothelial tumor cells, such as EZH2. In some examples, the specific binding agent preferentially binds to one or more of the identified ovarian endothelial cell tumor-associated molecules listed in any of Tables 1, 2, 4, 5 or a combination thereof that are downregulated in ovarian endothelial tumor cells (as indicated by a negative fold change in Table 1) to increase expression or activity of the one or more molecules. As a result, ovarian cancer in the subject is thereby reduced or eliminated.
Terms
The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms “a,” “an,” and “the” refer to one or more than one, unless the context clearly dictates otherwise. For example, the term “comprising a nucleic acid molecule” includes single or plural nucleic acid molecules and is considered equivalent to the phrase “comprising at least one nucleic acid molecule.” The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.
Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
Administration:
To provide or give a subject an agent, such as a chemotherapeutic agent, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
Agent:
Any protein, nucleic acid molecule, compound, small molecule, organic compound, inorganic compound, or other molecule of interest. Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject). In a particular example, a pharmaceutical agent (such as a siRNA to any of the genes listed in Tables 2 and Table 4) significantly reduces angiogenesis. A test agent is any substance, including, but not limited to, a protein (such as an antibody), nucleic acid molecule (such as a siRNA), organic compound, inorganic compound, or other molecule of interest. In particular examples, a test agent can permeate a cell membrane (alone or in the presence of a carrier).
Amplifying a Nucleic Acid Molecule:
To increase the number of copies of a nucleic acid molecule, such as a gene or fragment of a gene, for example a region of an ovarian endothelial cell tumor-associated gene. The resulting products are called amplification products.
An example of in vitro amplification is the polymerase chain reaction (PCR), in which a biological sample obtained from a subject (such as a sample containing ovarian cancer cells) is contacted with a pair of oligonucleotide primers, under conditions that allow for hybridization of the primers to a nucleic acid molecule in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule. Other examples of in vitro amplification techniques include quantitative real-time PCR, strand displacement amplification (see U.S. Pat. No. 5,744,311); transcription-free isothermal amplification (see U.S. Pat. No. 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see U.S. Pat. No. 5,427,930); coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); and NASBATM RNA transcription-free amplification (see U.S. Pat. No. 6,025,134).
A commonly used method for real-time quantitative polymerase chain reaction involves the use of a double stranded DNA dye (such as SYBR Green I dye). For example, as the amount of PCR product increases, more SYBR Green I dye binds to DNA, resulting in a steady increase in fluorescence. Another commonly used method is real-time quantitative TaqMan PCR (Applied Biosystems). The 5′ nuclease assay provides a real-time method for detecting only specific amplification products. The use of fluorogenic probes makes it possible to eliminate post-PCR processing for the analysis of probe degradation. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by Förster resonance energy transfer (FRET) through space. Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5′ end and the quencher at the 3′ end.
Angiogenesis:
A physiological process involving the growth of new blood vessels from pre-existing vessels. Angiogenesis can occur under normal physiological conditions such as during growth and development or wound healing (known as physiological angiogenesis) as well as pathological conditions such as in the transition of tumors from a dormant state to a malignant state (known as pathological angiogenesis). As used herein, pro-angiogenic genes are genes that facilitate angiogenesis, such as angiogenesis in an ovarian tumor.
The complex phenomenon of angiogenesis begins with degradation of the basement membrane by cellular proteases. This allows endothelial cells to penetrate and migrate (process known as cell motility) into the extracellular matrix and then proliferate. In the final stages of this process, the endothelial cells align themselves to form capillary or tubelike structures (process known as tube formation). These new structures then form a network that undergoes significant remodeling and rearrangement before fully functioning capillaries exist. Therefore, angiogenesis can be studied or identified by monitoring tube formation, cell motility, and/or cell proliferation.
Antibody:
A polypeptide ligand including at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as an ovarian endothelial cell tumor-associated molecule or a fragment thereof. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. In one example, an antibody specifically binds to one of the proteins listed in Tables 8 and 9.
This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab′ fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda and kappa. There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds RET will have a specific VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (such as different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).
References to “VH” or “VH” refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
A “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
A “polyclonal antibody” is an antibody that is derived from different B-cell lines. Polyclonal antibodies are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope. These antibodies are produced by methods known to those of skill in the art, for instance, by injection of an antigen into a suitable mammal (such as a mouse, rabbit or goat) that induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen which are then purified from the mammal's serum.
A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds an ovarian endothelial cell tumor-associated molecule.
A “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Pat. No. 5,585,089).
Array:
An arrangement of molecules, such as biological macromolecules (such as peptides or nucleic acid molecules) or biological samples (such as tissue sections), in addressable locations on or in a substrate. A “microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis.
The array of molecules (“features”) makes it possible to carry out a very large number of analyses on a sample at one time. In certain example arrays, one or more molecules (such as an oligonucleotide probe) will occur on the array a plurality of times (such as twice), for instance to provide internal controls. The number of addressable locations on the array can vary, for example from at least one, to at least 2, to at least 5, to at least 10, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, least 550, at least 600, at least 800, at least 1000, at least 10,000, or more. In particular examples, an array includes nucleic acid molecules, such as oligonucleotide sequences that are at least 15 nucleotides in length, such as about 15-40 nucleotides in length. In particular examples, an array includes oligonucleotide probes or primers which can be used to detect sensitive to ovarian endothelial cell tumor-associated molecule sequences, such as at least one of those listed in Table 1, such as at least 5, at least 7, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, or at least 1100 sequences listed in Table 1 (for example, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 175, 225, 275, 325, 350, 375, 450, 550, 650, 750, 850, 950, 1050 or 1149 of those listed). In an example, the array is a commercially available such as a U133 Plus 2.0 oligonucleotide array from Affymetrix (Affymetrix, Santa Clara, Calif.).
Within an array, each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array. The feature application location on an array can assume different shapes. For example, the array can be regular (such as arranged in uniform rows and columns) or irregular. Thus, in ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position. Often, ordered arrays are arranged in a symmetrical grid pattern, but samples could be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters). Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity). In some examples of computer readable formats, the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.
Protein-based arrays include probe molecules that are or include proteins, or where the target molecules are or include proteins, and arrays including nucleic acids to which proteins are bound, or vice versa. In some examples, an array contains antibodies to ovarian endothelial cell tumor-associated proteins, such as any combination of those listed in Table 1, such as at least 2, least 5, at least 7, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, or at least 1100 sequences listed in Table 1 (for example, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 175, 225, 275, 325, 350, 375, 450, 550, 650, 750, 850, 950, 1050 or 1149 of those listed).
Binding or Stable Binding:
An association between two substances or molecules, such as the hybridization of one nucleic acid molecule to another (or itself), the association of an antibody with a peptide, or the association of a protein with another protein or nucleic acid molecule. An oligonucleotide molecule binds or stably binds to a target nucleic acid molecule if a sufficient amount of the oligonucleotide molecule forms base pairs or is hybridized to its target nucleic acid molecule, to permit detection of that binding. “Preferentially binds” indicates that one molecule binds to another with high affinity, and binds to heterologous molecules at a low affinity.
Binding can be detected by any procedure known to one skilled in the art, such as by physical or functional properties of a target:oligonucleotide complex or a protein:antibody complex. For example, binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like.
Physical methods of detecting the binding of complementary strands of nucleic acid molecules, include but are not limited to, such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, dot blotting and light absorption detection procedures. For example, one method involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) and target disassociate from each other, or melt. In another example, the method involves detecting a signal, such as a detectable label, present on one or both nucleic acid molecules (or antibody or protein as appropriate).
The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (Tm) at which 50% of the oligomer is melted from its target. A higher (Tm) means a stronger or more stable complex relative to a complex with a lower (Tm).
Biological Activity:
An expression describing the beneficial or adverse effects of an agent on living matter. When the agent is a complex chemical mixture, this activity is exerted by the substance's active ingredient or pharmacophore, but can be modified by the other constituents. Activity is generally dosage-dependent and it is not uncommon to have effects ranging from beneficial to adverse for one substance when going from low to high doses. In one example, a specific binding agent significantly reduces the biological activity of the one or more ovarian endothelial cell tumor-associated molecules that is upregulated in ovarian endothelial tumor cells (such as those listed in Tables 2 and 4) which reduces or eliminates ovarian cancer, such as by reducing or inhibiting angiogenesis. In some examples, a specific binding agent significantly increases the biological activity of one or more ovarian endothelial cell tumor-associated molecules that is downregulated in ovarian endothelial tumor cells (such as those listed in Table 3).
Cancer:
The “pathology” of cancer includes all phenomena that compromise the well-being of the subject. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. “Metastatic disease” refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.
Chemotherapeutic Agent or Chemotherapy:
Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is an agent of use in treating ovarian cancer, such as papillary serous ovarian cancer. In one example, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer and Berkery. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer Knobf, and Durivage (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Chemotherapeutic agents used for treating ovarian cancer include, but are not limited to, carboplatin, cisplatin, paclitaxel, docetaxel, doxorubicin, epirubicin, topotecan, irinotecan, gemcitabine, iazofurine, gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide, methotrexate, fluorouracil, mitoxantrone and vinorelbine. Combination chemotherapy is the administration of more than one agent (such as more than one chemotherapeutic agent) to treat cancer.
Chrondroitin Sulfate Proteoglycan 2 (CSPG2):
An extracellular matrix component of the vitreous gel that has been reported to be an anti-cell adhesive. In particular examples, expression of CSPG2 is increased in ovarian cancer endothelial cells. The term CSPG2 includes any CSPG2 gene, cDNA, mRNA, or protein from any organism and that is CSPG2 and is expressed and in some examples overexpressed in ovarian cancer endothelial cells.
Nucleic acid and protein sequences for CSPG2 are publicly available. For example, GenBank Accession Nos.: NM—004385 and BC096495 disclose CSPG2 nucleic acid sequences, and GenBank Accession Nos.: AAH50524, NP—004376, and AAH96495 disclose CSPG2 protein sequences, all of which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, CSPG2 includes a full-length wild-type (or native) sequence, as well as CSPG2 allelic variants that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, CSPG2 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No. AAH50524, NP—004376, or AAH96495. In other examples, CSPG2 has a sequence that hybridizes to AFFYMETRIX® Probe ID No. 204619_s_at and 221731_x_a and retains CSPG2 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Complementarity and Percentage Complementarity:
Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide molecule remains detectably bound to a target nucleic acid sequence (such as an ovarian endothelial cell tumor-associated molecule) under the required conditions.
Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, that is, the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands. For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.
In the present disclosure, “sufficient complementarity” means that a sufficient number of base pairs exist between an oligonucleotide molecule and a target nucleic acid sequence (such as a ovarian endothelial cell tumor-associated molecule, for example any of the genes listed in Table 1) to achieve detectable binding. When expressed or measured by percentage of base pairs formed, the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary. In general, sufficient complementarity is at least about 50%, for example at least about 75% complementarity, at least about 90% complementarity, at least about 95% complementarity, at least about 98% complementarity, or even at least about 100% complementarity.
A thorough treatment of the qualitative and quantitative considerations involved in establishing binding conditions that allow one skilled in the art to design appropriate oligonucleotides for use under the desired conditions is provided by Beltz et al. Methods Enzymol. 100:266-285, 1983, and by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
Contacting:
Placement in direct physical association, including both a solid and liquid form. Contacting can occur in vitro, for example, with isolated cells or in vivo by administering to a subject.
Decrease:
To reduce the quality, amount, or strength of something. In one example, a therapy decreases a tumor (such as the size of a tumor, the growth of a tumor, number of tumors, the metastasis of a tumor, or combinations thereof), or one or more symptoms associated with a tumor, for example as compared to the response in the absence of the therapy (such as a therapy administered to affect tumor size by inhibiting angiogenesis via administration of a binding agent capable of binding to one or more of the ovarian endothelial cell tumor-associated markers listed in Tables 1 through 5 that is involved in promoting angiogenesis, such as by inhibiting an ovarian endothelial cell tumor-associated marker that is upregulated in ovarian endothelial tumor cells or by increasing activity of an ovarian endothelial cell tumor-associated marker that is downregulated in ovarian endothelial tumor cells). In a particular example, a therapy decreases the size of a tumor, the growth of a tumor, the number of tumors, the metastasis of a tumor, or combinations thereof, subsequent to the therapy, such as a decrease of at least 10%, at least 20%, at least 50%, or even at least 90%. Such decreases can be measured using the methods disclosed herein.
Determining Expression of a Gene Product:
Detection of a level expression in either a qualitative or quantitative manner.
Diagnosis:
The process of identifying a disease by its signs, symptoms and results of various tests. The conclusion reached through that process is also called “a diagnosis.” Forms of testing commonly performed include blood tests, medical imaging, urinalysis, and biopsy.
DNA (Deoxyribonucleic Acid):
A long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached. Triplets of nucleotides, referred to as codons, in DNA molecules code for amino acid in a polypeptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed.
Differential Expression:
A difference, such as an increase or decrease, in the conversion of the information encoded in a gene (such as an ovarian endothelial cell tumor-associated molecule) into messenger RNA, the conversion of mRNA to a protein, or both. In some examples, the difference is relative to a control or reference value, such as an amount of gene expression that is expected in a subject who does not have ovarian cancer or in a normal (non-cancerous) endothelial cell sample. Detecting differential expression can include measuring a change in gene expression.
Downregulated or Inactivation:
When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in a decrease in production of a gene product. A gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, gene downregulation or deactivation includes processes that decrease transcription of a gene or translation of mRNA. Examples of genes whose expression is downregulated in ovarian tumor endothelial cells can be found in Table 1 (indicated by a negative fold change, such as TLOC1 and HS6ST2) and Table 3 (such as PLN, SELE, GREB1, OGN and LCXD3).
Examples of processes that decrease transcription include those that facilitate degradation of a transcription initiation complex, those that decrease transcription initiation rate, those that decrease transcription elongation rate, those that decrease processivity of transcription and those that increase transcriptional repression. Gene downregulation can include reduction of expression above an existing level. Examples of processes that decrease translation include those that decrease translational initiation, those that decrease translational elongation and those that decrease mRNA stability.
Gene downregulation includes any detectable decrease in the production of a gene product. In certain examples, production of a gene product decreases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal endothelial cell). In one example, a control is a relative amount of gene expression or protein expression in a biological sample taken from a subject who does not have ovarian cancer.
Endothelial Cell:
Cells that line the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. For example, endothelial cells line the entire circulatory system. Further, both blood and lymphatic capillaries are composed of a single layer of endothelial cells.
Epidermal Growth Factor-like Domain Multiple 6 (EGFL6):
A member of the epidermal growth factor (EGF) repeat superfamily of genes known to encode proteins that govern cellular proliferative responses. EGFL6 has been identified as a possible regulator of cell cycle and oncogenesis.
In particular examples, expression of EGFL6 is increased in ovarian cancer endothelial cells. The term EGFL6 includes any EGFL6 gene, cDNA, mRNA, or protein from any organism and that is EGFL6 and is expressed and in some examples overexpressed in ovarian cancer endothelial cells.
Nucleic acid and protein sequences for EGFL6 are publicly available. For example, GenBank Accession Nos.: NM—015507, NM—019397 and BC038587 disclose EGFL6 nucleic acid sequences, and GenBank Accession Nos.: AAQ88699, CAM23572, and AAF27812 disclose EGFL6 protein sequences, all of which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, EGFL6 includes a full-length wild-type (or native) sequence, as well as EGFL6 allelic variants that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, EGFL6 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No. AAQ88699, CAM23572, or AAF27812. In other examples, EGFL6 has a sequence that hybridizes to Affymetrix Probe ID No. 219454_at and retains EGFL6 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Expression:
The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.
The expression of a nucleic acid molecule can be altered relative to a normal (wild type) nucleic acid molecule. Alterations in gene expression, such as differential expression, include but are not limited to: (1) overexpression; (2) underexpression; or (3) suppression of expression. Alternations in the expression of a nucleic acid molecule can be associated with, and in fact cause, a change in expression of the corresponding protein. Specific examples of ovarian endothelial cell tumor-associated molecules that are up-regulated in ovarian tumor endothelial cells are provided in Tables 2 and 4. Specific examples of ovarian endothelial cell tumor-associated molecules that are down-regulated in ovarian tumor endothelial cells are listed in Table 3. For example, EZH2, EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, and PLXDC1 are upregulated or increased in expression in ovarian tumor endothelial cells, while TLOC1 and HS6ST2 are downregulated or decreased in expression in such cells.
Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (wild type) situation. This includes but is not necessarily limited to: (1) a mutation in the protein such that one or more of the amino acid residues is different; (2) a short deletion or addition of one or a few (such as no more than 10-20) amino acid residues to the sequence of the protein; (3) a longer deletion or addition of amino acid residues (such as at least 20 residues), such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount of the protein compared to a control or standard amount; (5) expression of a decreased amount of the protein compared to a control or standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporally regulated expression of the protein (such that the protein is expressed when it normally would not be, or alternatively is not expressed when it normally would be); (8) alteration in stability of a protein through increased longevity in the time that the protein remains localized in a cell; and (9) alteration of the localized (such as organ or tissue specific or subcellular localization) expression of the protein (such that the protein is not expressed where it would normally be expressed or is expressed where it normally would not be expressed), each compared to a control or standard.
Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have cancer, such as ovarian cancer) as well as laboratory values, even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.
Laboratory standards and values may be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.
Gene Expression Profile (or Fingerprint):
Differential or altered gene expression can be detected by changes in the detectable amount of gene expression (such as cDNA or mRNA) or by changes in the detectable amount of proteins expressed by those genes. A distinct or identifiable pattern of gene expression, for instance a pattern of high and low expression of a defined set of genes or gene-indicative nucleic acids such as ESTs; in some examples, as few as one or two genes provides a profile, but more genes can be used in a profile, for example at least 3, at least 4, at least 5, at least 6, at least 10, at least 20, at least 25, at least 30, at least 50, at least 80, at least 100, at least 190, at least 200, at least 300, at least 400, at least 500, at least 550, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100 or more of those listed in any of Tables 1-5. A gene expression profile (also referred to as a fingerprint) can be linked to a tissue or cell type (such as ovarian cancer cell), to a particular stage of normal tissue growth or disease progression (such as advanced ovarian cancer), or to any other distinct or identifiable condition that influences gene expression in a predictable way. Gene expression profiles can include relative as well as absolute expression levels of specific genes, and can be viewed in the context of a test sample compared to a baseline or control sample profile (such as a sample from a subject who does not have ovarian cancer or normal endothelial cells). In one example, a gene expression profile in a subject is read on an array (such as a nucleic acid or protein array). For example, a gene expression profile is performed using a commercially available array such as a Human Genome U133 2.0 Plus Microarray from AFFYMETRIX® (AFFYMETRIX®, Santa Clara, Calif.).
Homeodomain-Only Protein, Transcript Variant 2 (HOP):
A transcriptional repressor that modulates serum response factor-dependent cardiac-specific gene expression and cardiac development. In particular examples, expression of HOP is increased in ovarian cancer endothelial cells. The term HOP includes any HOP gene, cDNA, mRNA, or protein from any organism and that is HOP and is expressed and in some examples overexpressed in ovarian cancer endothelial cells.
Nucleic acid and protein sequences for HOP are publicly available. For example, GenBank Accession Nos.: NM—139211, XM—001083738, and XM—001137349 disclose HOP nucleic acid sequences, and GenBank Accession Nos.: AAH14225, NP—631958, and NP—631957 disclose HOP protein sequences, all of which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, HOP includes a full-length wild-type (or native) sequence, as well as HOP allelic variants that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, HOP has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No.: AAH14225, NP—631958, or NP—631957. In other examples, HOP has a sequence that hybridizes to AFFYMETRIX® Probe ID No. 211597_s_at and retains HOP activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Hybridization:
To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). The following is an exemplary set of hybridization conditions and is not limiting:
Very High Stringency (Detects Sequences that Share at Least 90% Identity)
Hybridization: 5×SSC at 65° C. for 16 hours
Wash twice: 2×SSC at room temperature (RT) for 15 minutes each
Wash twice: 0.5×SSC at 65° C. for 20 minutes each
High Stringency (Detects Sequences that Share at Least 80% Identity)
Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours
Wash twice: 2×SSC at RT for 5-20 minutes each
Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each
Low Stringency (Detects Sequences that Share Greater than 50% Identity)
Hybridization: 6×SSC at RT to 55° C. for 16-20 hours
Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.
Inhibitor:
Any chemical compound, nucleic acid molecule, peptide or polypeptide such as an antibody or RNAi that can reduce activity of a gene product or interfere with expression of a gene, respectively. In some examples, an inhibitor can reduce or inhibit the activity of a protein that is encoded by a gene either directly or indirectly. Direct inhibition can be accomplished, for example, by binding to a protein and thereby preventing the protein from binding an intended target, such as a receptor. Indirect inhibition can be accomplished, for example, by binding to a protein's intended target, such as a receptor or binding partner, thereby blocking or reducing activity of the protein. In some examples, an inhibitor of the disclosure can inhibit a gene by reducing or inhibiting expression of the gene, inter alia by interfering with gene expression (transcription, processing, translation, post-translational modification), for example, by interfering with the gene's mRNA and blocking translation of the gene product or by post-translational modification of a gene product, or by causing changes in intracellular localization.
Isolated:
An “isolated” biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been “isolated” include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. For example, an isolated serous papillary ovarian cancer cell is one that is substantially separated from other ovarian cell subtypes, such as endometrioid, clear cell or mucinous subtypes.
Label:
An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). In a particular example, a label is conjugated to a binding agent that specifically binds to one or more of the ovarian endothelial cell tumor-associated molecules disclosed in Tables 1 through 5 to allow for the detection/screening for angiogenesis and/or the presence of a tumor in a subject.
Malignant:
Cells that have the properties of anaplasia invasion and metastasis.
Mammal:
This term includes both human and non-human mammals. Examples of mammals include, but are not limited to: humans and veterinary and laboratory animals, such as pigs, cows, goats, cats, dogs, rabbits and mice.
Neoplasm:
Abnormal growth of cells.
Normal Cell:
Non-tumor cell, non-malignant, uninfected cell.
Nucleic Acid Array:
An arrangement of nucleic acids (such as DNA or RNA) in assigned locations on a matrix, such as that found in cDNA arrays, or oligonucleotide arrays, such as those listed in Tables 1-5.
Nucleic Acid Molecules Representing Genes:
Any nucleic acid, for example DNA (intron or exon or both), cDNA, or RNA (such as mRNA), of any length suitable for use as a probe or other indicator molecule, and that is informative about the corresponding gene.
Nucleic Acid Molecules:
A deoxyribonucleotide or ribonucleotide polymer including, without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA. The nucleic acid molecule can be double-stranded or single-stranded. Where single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. In addition, nucleic acid molecule can be circular or linear.
The disclosure includes isolated nucleic acid molecules that include specified lengths of an ovarian endothelial cell tumor-associated molecule nucleotide sequence, for sequences for genes listed in Tables 1 through 4. Such molecules can include at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 consecutive nucleotides of these sequences or more, and can be obtained from any region of a ovarian endothelial cell tumor-associated molecule.
Oligonucleotide:
A plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.
Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 nucleotides, for example at least 8, at least 10, at least 15, at least 20, at least 21, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100 or even at least 200 nucleotides long, or from about 6 to about 50 nucleotides, for example about 10-25 nucleotides, such as 12, 15 or 20 nucleotides.
Oligonucleotide Probe:
A short sequence of nucleotides, such as at least 8, at least 10, at least 15, at least 20, at least 21, at least 25, or at least 30 nucleotides in length, used to detect the presence of a complementary sequence by molecular hybridization. In particular examples, oligonucleotide probes include a label that permits detection of oligonucleotide probe:target sequence hybridization complexes, such as with an ovarian endothelial cell tumor-associated molecule listed in Tables 1-5.
Ovarian Cancer: A
malignant ovarian neoplasm (an abnormal growth located on the ovaries). Cancer of the ovaries includes ovarian carcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid tumors, celioblastoma, clear cell carcinoma, unclassified carcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, and malignant teratoma. The most common type of ovarian cancer is papillary serous carcinoma.
Surgery is an exemplary treatment for ovarian cancer and can be necessary for diagnosis. The type of surgery depends upon how widespread the cancer is when diagnosed (the cancer stage), as well as the type and grade of cancer. The surgeon may remove one (unilateral oophorectomy) or both ovaries (bilateral oophorectomy), the fallopian tubes (salpingectomy), and the uterus (hysterectomy). For some very early tumors (stage 1, low grade or low-risk disease), only the involved ovary and fallopian tube will be removed (called a “unilateral salpingo-oophorectomy,” USO), especially in young females who wish to preserve their fertility. In advanced disease as much tumor as possible is removed (debulking surgery). In cases where this type of surgery is successful, the prognosis is improved compared to subjects where large tumor masses (more than 1 cm in diameter) are left behind.
Chemotherapy is often used after surgery to treat any residual disease. At present systemic chemotherapy often includes a platinum derivative with a taxane as a method of treating advanced ovarian cancer. Chemotherapy is also used to treat subjects who have a recurrence.
Ovarian Endothelial Cell Tumor-Associated (or Related) Molecule:
A molecule whose expression is altered in ovarian tumor endothelial cells. Such molecules include, for instance, nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins. Specific genes include those listed in Tables 1 through 5. Thus, the presence of the respective ovarian endothelial cell tumor-associated molecules can be used to diagnose, or determine the prognosis of, an ovarian tumor in a subject.
In an example, an ovarian endothelial cell tumor-associated molecule is any molecule listed in Tables 1 through 5. Specific examples of ovarian endothelial cell tumor-associated molecules that are up-regulated in ovarian tumor endothelial cells are provided in Tables 2 and 4. Specific examples of ovarian endothelial cell tumor-associated molecules that are down-regulated in ovarian tumor endothelial cells are listed in Table 3. As illustrated in Table 4, a number of the identified ovarian cell tumor-associated molecules are related to cell proliferation, tube formation and cell motility.
Ovarian endothelial cell tumor-associated molecules can be involved in or influenced by cancer in different ways, including causative (in that a change in a ovarian endothelial cell tumor-associated molecule leads to development of or progression of ovarian cancer) or resultive (in that development of or progression of ovarian cancer causes or results in a change in the ovarian endothelial cell tumor-associated molecule).
Pharmaceutically Acceptable Carriers:
The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic agents, such as one or more compositions that include a binding agent that specifically binds to at least one of the disclosed ovarian endothelial cell tumor-associated molecules.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.
Plexin Domain Containing 1 (PLXDC1):
A large transmembrane receptor. In vitro, plexin-C1 has been shown to bind the GPI-anchored semaphorin Sema7A and the soluble viral semaphorins SemaVA (A39R) and SemaVB (AHV). Plexin C1 engagement by SemaVA inhibits integrin-mediated dendritic cell adhesion and chemotaxis in vitro, suggesting a role for plexin C1 in dendritic cell migration.
In an example, expression of PLXDL1 is increased in ovarian tumor endothelial cells. The term PLXDC1 includes any plexin C1 gene, cDNA, mRNA, or protein from any organism and that is a PLXDC1 and is expressed and in some examples overexpressed in ovarian tumor endothelial cells.
Exemplary nucleic acid and protein sequences for PLXDC1 are publicly available. For example, GenBank Accession Nos.: NM—018797, XM—622776, AB208934, and NM—005761 disclose PLXDC1 nucleic acid sequences and GenBank Accession Nos.: NP—061267, XP—622776, BAD92171, and NP—005752 disclose PLXDC1 protein sequences, all of which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, a PLXDC1 sequence includes a full-length wild-type (or native) sequence, as well as PLXDC1 allelic variants, fragments, homologs or fusion sequences that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, PLXDC1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No.: NP—061267, XP—622776, BAD92171, or NP—005752. In other examples, a PLXDC1 has a sequence that hybridizes to AFFYMETRIX® Probe ID No. 214081_at and retains PLXDC1 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Polymerase Chain Reaction (PCR):
An in vitro amplification technique that increases the number of copies of a nucleic acid molecule (for example, a nucleic acid molecule in a sample or specimen). In an example, a biological sample collected from a subject (e.g., with ovarian cancer) is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of a PCR can be characterized by methods known in the art such as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques.
Primers:
Short nucleic acid molecules, for instance DNA oligonucleotides 10-100 nucleotides in length, such as about 15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand (such as a gene listed in Table 1) by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand. Primer pairs can be used for amplification of a nucleic acid sequence, such as by PCR or other nucleic acid amplification methods known in the art.
Methods for preparing and using nucleic acid primers are described, for example, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), and Innis et al. (PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc., San Diego, Calif., 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). One of ordinary skill in the art will appreciate that the specificity of a particular primer increases with its length. Thus, for example, a primer including 30 consecutive nucleotides of an ovarian endothelial cell tumor-associated molecule will anneal to a target sequence, such as another homolog of the designated endothelial cell tumor-associated protein, with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, primers can be selected that include at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of an ovarian endothelial cell tumor-associated nucleotide sequence.
Prognosis:
A prediction of the course of a disease, such as ovarian cancer. The prediction can include determining the likelihood of a subject to develop aggressive, recurrent disease, to survive a particular amount of time (e.g., determine the likelihood that a subject will survive 1, 2, 3 or 5 years), to respond to a particular therapy (e.g., chemotherapy), or combinations thereof.
Purified:
The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell. For example, a preparation of a protein is purified such that the protein represents at least 50% of the total protein content of the preparation. Similarly, a purified oligonucleotide preparation is one in which the oligonucleotide is more pure than in an environment including a complex mixture of oligonucleotides.
Recombinant:
A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.
Sample (or Biological Sample):
A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. In one example, a sample includes an ovarian cancer tissue biopsy.
Sensitivity:
A measurement of activity, such as biological activity, of a molecule or a collection of molecules in a given condition. In an example, sensitivity refers to the activity of an agent, such as a binding agent that preferentially binds to one or more ovarian endothelial cell tumor-associated molecules, to alter the growth, development or progression of a disease, such as ovarian cancer. In certain examples, sensitivity or responsiveness can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size; (4) inhibition (such as reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (such as reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; (9) decreased mortality at a given point of time following treatment; and/or (10) reducing or inhibiting angiogenesis.
Sequence Identity/Similarity:
The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1154 nucleotides is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15÷20*100=75).
For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the proteins listed in Table 1.
When aligning short peptides (fewer than around 30 amino acids), the alignment is be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity with the proteins listed in Table 1. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.
One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with the genes listed in Table 1 as determined by this method. An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
One of skill in the art will appreciate that the particular sequence identity ranges are provided for guidance only; it is possible that strongly significant homologs could be obtained that fall outside the ranges provided.
Short interfering RNA (siRNA):
A double stranded nucleic acid molecule capable of RNA interference or “RNAi.” (See, for example, Bass Nature 411: 428-429, 2001; Elbashir et al., Nature 411: 494-498, 2001; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914.) As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides having RNAi capacity or activity. In an example, an siRNA molecule is one that reduces or inhibits the biological activity or expression of one or more ovarian endothelial cell tumor-associated molecules disclosed in Tables 1, 2, 4 or 5 that are upregulated in ovarian tumor endothelial cells, such as EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, PLXDC1, EZH2, the Notch ligand Jagged1 or PTK2.
Specific Binding Agent:
An agent that binds substantially or preferentially only to a defined target (for example, those listed in Table 1), such as a protein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinant vector or a small molecule. In an example, a “specific binding agent” is capable of binding to at least one of the disclosed ovarian endothelial cell tumor-associated molecules. Thus, a RNA-specific binding agent binds substantially only to the defined RNA, or to a specific region within the RNA. For example, a “specific binding agent” includes a siRNA that binds substantially to a specified RNA.
A protein-specific binding agent binds substantially only the defined protein, or to a specific region within the protein. For example, a “specific binding agent” includes antibodies and other agents that bind substantially to a specified polypeptide. The antibodies can be monoclonal or polyclonal antibodies that are specific for the polypeptide, as well as immunologically effective portions (“fragments”) thereof. The determination that a particular agent binds substantially only to a specific polypeptide may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).
Stanniocalcin 1 (STC1):
A hormone that plays a role in calcium regulation, phosphate homeostasis and cell metabolism. In particular examples, expression of STC1 is increased in ovarian tumor endothelial cells. The term STC1 includes any STC1 gene, cDNA, mRNA, or protein from any organism and that is STC1 and is expressed or overexpressed in some examples in ovarian tumor endothelial cells.
Nucleic acid and protein sequences for STC1 are publicly available. For example, GenBank Accession Nos.: NM—009285, NM—00003155, and NM—031123 disclose STC1 nucleic acid sequences, and GenBank Accession Nos.: AAH21425, NP—112385, and NP—033311 disclose STC1 protein sequences, all of which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, STC1 includes a full-length wild-type (or native) sequence, as well as STC1 allelic variants that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, STC1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No.: AAH21425, NP—112385, or NP—033311. In other examples, STC1 has a sequence that hybridizes to AFFYMETRIX® Probe ID No. 230746_s_at, 204595_s_at, and 204597_x_at and retains STC1 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Subject:
Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.
Target Sequence:
A sequence of nucleotides located in a particular region in the human genome that corresponds to a desired sequence, such as an ovarian endothelial cell tumor-associated sequence. The target can be for instance a coding sequence; it can also be the non-coding strand that corresponds to a coding sequence. Examples of target sequences include those sequences associated with ovarian tumor endothelial cells, such as any of those listed in Tables 1 through 5.
Therapeutically Effective Amount:
An amount of a composition that alone, or together with an additional therapeutic agent(s) (for example a chemotherapeutic agent), induces the desired response (e.g., treatment of a tumor). The preparations disclosed herein are administered in therapeutically effective amounts.
In one example, a desired response is to decrease ovarian tumor size or metastasis in a subject to whom the therapy is administered. Tumor metastasis does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease metastasis by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of the tumor), as compared to metastasis in the absence of the composition.
In particular examples, it is an amount of the therapeutic agent conjugated to the specific binding agent effective to decrease a number of ovarian cancer cells, such as in a subject to whom it is administered, for example a subject having one or more ovarian carcinomas. The cancer cells do not need to be completely eliminated for the composition to be effective. For example, a composition can decrease the number of cancer cells or growth of such cells by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable cancer cells), as compared to the number of cancer cells in the absence of the composition.
In other examples, it is an amount of the specific binding agent for one or more of the disclosed ovarian endothelial cell tumor-associated molecules capable of reducing angiogenesis by least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable angiogenesis) by the specific binding agent, or both, effective to decrease the metastasis of a tumor.
A therapeutically effective amount of a specific binding agent for at least one of the disclosed ovarian endothelial cell tumor-associated molecules, or cancer cells lysed by a therapeutic molecule conjugated to the agent, can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. For example, a therapeutically effective amount of such agent can vary from about 1 μg-10 mg per 70 kg body weight if administered intravenously and about 10 μg-100 mg per 70 kg body weight if administered intratumorally.
Tissue:
A plurality of functionally related cells. A tissue can be a suspension, a semi-solid, or solid. Tissue includes cells collected from a subject such as the ovaries.
Treating a Disease:
“Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as a sign or symptom of ovarian cancer. Treatment can also induce remission or cure of a condition, such as ovarian cancer. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient.
Tumor:
All neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. In an example, a tumor is an ovarian tumor.
Tumor-Necrosis Factor, Alpha-Induced Protein 6 (TNFAIP6):
A protein capable of regulating the expression of various molecules involved in the control of inflammation. In particular examples, expression of TNFAIP6 is increased in ovarian cancer endothelial cells. The term TNFAIP6 includes any TNFAIP6 gene, cDNA, mRNA, or protein from any organism and that is TNFAIP6 and is expressed or overexpressed in some examples in ovarian cancer endothelial cells.
Nucleic acid and protein sequences for TNFAIP6 are publicly available. For example, GenBank Accession Nos.: NM—007115, BC021155 and NM—009398 disclose TNFAIP6 nucleic acid sequences, and GenBank Accession Nos.: AAH21155, NP—009046 and NP—033424 disclose TNFAIP6 protein sequences, all of which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, TNFAIP6 includes a full-length wild-type (or native) sequence, as well as TNFAIP6 allelic variants that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as suppression of vascular growth. In certain examples, TNFAIP6 has at least 80% sequence identity, for example, at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No.: AAH21155, NP—009046 or NP—033424. In other examples, TNFAIP6 has a sequence that hybridizes to AFFYMETRIX® Probe ID No. 206026_s_at and retains TNFAIP6 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Twist Homologue 1 (TWIST1):
Overexpression of TWIST1 has been reported to play a role in destabilizing the genome, thus promoting chromosomal instability. For example, TWIST1 is capable of inhibiting chrondrogenesis. TWIST1 protein has also been noted to be involved in the regulation of tumor necrosis factor alpha production by antiinflammatory factors and pathways. In particular examples, expression of TWIST1 is increased in ovarian cancer endothelial cells. The term TWIST1 includes any TWIST1 gene, cDNA, mRNA, or protein from any organism and that is TWIST1 and is expressed or overexpressed in some examples in ovarian cancer endothelial cells.
Nucleic acid and protein sequences for TWIST1 are publicly available. For example, GenBank Accession Nos.: NM—000474, NM—053530 and XM—001076553 and disclose TWIST1 nucleic acid sequences, and GenBank Accession Nos.: NP—000465 and ABM87769 disclose TWIST1 protein sequences, all of which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, TWIST1 includes a full-length wild-type (or native) sequence, as well as TWIST1 allelic variants that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, TWIST1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No.: NP—000465 or ABM87769. In other examples, TWIST1 has a sequence that hybridizes to AFFYMETRIX® Probe ID No. 206026_s_at and retains TWIST1 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Under Conditions Sufficient for:
A phrase that is used to describe any environment that permits the desired activity. In one example, includes administering a test agent to an ovarian cancer cell or a subject sufficient to allow the desired activity. In particular examples, the desired activity is altering the activity (such as the expression) of an ovarian endothelial cell tumor-associated molecule.
Unit Dose:
A physically discrete unit containing a predetermined quantity of an active material calculated to individually or collectively produce a desired effect, such as a therapeutic effect. A single unit dose or a plurality of unit doses can be used to provide the desired effect, such as treatment of a tumor, for example a metastatic tumor. In one example, a unit dose includes a desired amount of an agent that decreases or inhibits angiogenesis.
Upregulated or Activation:
When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in an increase in production of a gene product. A gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, gene upregulation or activation includes processes that increase transcription of a gene or translation of mRNA. Specific examples of ovarian endothelial cell tumor-associated molecules that are up-regulated in ovarian tumor endothelial cells are provided in Tables 2 and 4. For example, EZH2, EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, and PLXDC1 are upregulated or increased in expression in ovarian tumor endothelial cells.
Examples of processes that increase transcription include those that facilitate formation of a transcription initiation complex, those that increase transcription initiation rate, those that increase transcription elongation rate, those that increase processivity of transcription and those that relieve transcriptional repression (for example by blocking the binding of a transcriptional repressor). Gene upregulation can include inhibition of repression as well as stimulation of expression above an existing level. Examples of processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability.
Gene upregulation includes any detectable increase in the production of a gene product. In certain examples, production of a gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal endothelial cell). In one example, a control is a relative amount of gene expression in a biological sample, such as in an ovarian tissue biopsy obtained from a subject that does not have ovarian cancer.
Vasohibin 1 (VASH1):
a protein that is expressed in a variety of tissues and inhibits functions relevant to neovascularization (migration, proliferation, and network formation by endothelial cells). Vasohibin also inhibits angiogenesis in vivo. The unglycosylated protein (42 kDa) does not contain a classical secretory secretion sequence and appears in the medium as a protein of 30 kDa, suggesting proteolytic processing during secretion. In particular examples, VASH1 is regulated by EZH2 wherein EZH2 binds to the VASH1 promoter and decreases or inhibits VASH1 anti-angiogenesis activity. The term VASH1 includes any VASH1 gene, cDNA, mRNA, or protein from any organism and that is VASH1 and is expressed or overexpressed in some examples in ovarian cancer endothelial cells.
Nucleic acid and protein sequences for VASH1 are publicly available. For example, GenBank Accession Nos.: NP—055724 (human); NP—796328 (mouse); and NP—659128 disclose VASH1 amino acid sequences which are incorporated by reference as provided by GenBank on Aug. 14, 2009. Further, GenBank Accession Nos.: NM—014909 (human) and NM—177354 (mouse) disclose nucleic acid sequences which are incorporated by reference as provided by GenBank on Aug. 14, 2009.
In one example, vasohibin includes a full-length wild-type (or native) sequence, as well as VASH1 allelic variants that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, VASH1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to GenBank Accession No.: NP—055724; NP—796328 or NP—659128 and retains VASH1 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Zeste Homologue 2 (EZH2):
A member of the polycomb group of genes that has been reported to be involved in cell cycle regulation EZH2, a component of the polycomb repressive complex 2 (PRC2), has intrinsic histone methyl transferase (HMTase) activity and has been implicated in the progression and metastasis of several cancers. EZH2 is also a transcriptional repressor that has multiple targets, including anti-angiogenic, pro-apoptotic, and tumor suppressor genes. In particular examples, expression of EZH2 is increased in ovarian cancer endothelial cells. In one example, expression of EZH2 is an indicator of poor prognosis. The term EZH2 includes any EZH2 gene, cDNA, mRNA, or protein from any organism and that is EZH2 and is expressed or overexpressed in some examples in ovarian cancer endothelial cells.
Nucleic acid and protein sequences for EZH2 are publicly available. For example, GenBank Accession Nos.: NM—004456 and AY519465.1 disclose EZH2 nucleic acid sequences, and GenBank Accession No. AAS09975 discloses a EZH2 protein sequence, which are incorporated by reference as provided by GenBank on Feb. 14, 2007.
In one example, EZH2 includes a full-length wild-type (or native) sequence, as well as EZH2 allelic variants, fragments, homologs or fusion sequences that retain the ability to be expressed in ovarian tumor endothelial cells and/or modulate ovarian tumor endothelial cells, such as increase vascular growth. In certain examples, EZH2 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to AAS09975. In other examples, EZH2 has a sequence that hybridizes to AFFYMETRIX® Probe ID No. 203358_s_at and retains EZH2 activity (such as the capability to be expressed in ovarian tumor endothelial cells and/or modulate tumor and/or vascular growth).
Additional terms commonly used in molecular genetics can be found in Benjamin Lewin, Genes V published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Methods of Treatment
It is shown herein that ovarian cancer is associated with differential expression of ovarian endothelial cell tumor-associated molecules. For example, the disclosed gene expression profile has identified ovarian endothelial cell tumor-associated molecules. Based on these observations, methods of treatment to reduce or eliminate ovarian cancer are disclosed. For example, the method can include inhibiting the expression or biological activity of at least one of the ovarian endothelial cell tumor associated molecules from Tables 1, 2, 4, and/or 5 that are upregulated in ovarian tumor cells or increasing the expression or biological activity of at least one of the ovarian endothelial cell tumor associated molecules from Tables 1, 3 and/or 5 that are downregulated in ovarian tumor cells or combinations thereof. As used herein, “inhibit” does not require 100% inhibition of expression or activity. For example, a substantial reduction may be adequate, such as reduction in expression or activity of at least 20%, at least 30%, at least 50%, at least 75%, or at least 95% may be sufficient to obtain desired therapeutic results. In some examples, an “increase” in expression or activity is an increase of at least 20%, at least 30%, at least 50%, at least 75%, or at least 95%. In some embodiments, the subject is a human, but the subject can alternatively be a veterinary or laboratory subject. In some embodiments, the ovarian cancer is papillary serous ovarian cancer.
Methods are disclosed herein for treating an ovarian tumor, such as ovarian cancer. In one example, the method includes administering a therapeutically effective amount of a composition to a subject. The composition can include a binding agent that is specific for one of the ovarian endothelial cell tumor-associated molecules listed in any of Tables 1, 2, 4 or 5 that are upregulated in ovarian tumor cells. Administration of such compounds decreases the expression or activity of the molecule that is undesirably upregulated in ovarian cancer cells. The molecules in Tables 1, 2, 4, or 5 include, for instance, nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins. Specific genes include those listed in Tables 1, 2, 4 or 5 as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby) whose expression is upregulated in response to an ovarian tumor, such as ovarian cancer.
In particular examples, the specific binding agent is an inhibitor such as a siRNA or an antibody to one of the disclosed ovarian endothelial cell tumor-associated molecules that is upregulated in ovarian tumor cells. For example, the specific binding agent can be a siRNA that interferes with mRNA expression of one of the disclosed ovarian endothelial cell tumor-associated molecules that are involved in angiogenesis, such as a molecule involved in regulating cell motility, cell proliferation or tube formation, thereby inhibiting cell motility, cell proliferation or tube formation. For example, the specific binding agent can be a siRNA that inhibits the expression of PTK2, EZH2 or Jagged1. In other particular examples, ovarian tumor growth is reduced or inhibited by administering a specific binding agent to inhibit or reduce the expression or production of EZH2, EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, and PLXDC1. In additional examples, a composition includes at least two specific binding agents such as two specific siRNAs that each bind to their respective ovarian endothelial cell tumor-associated nucleotide sequences and inhibit ovarian tumor growth in a subject. In some examples, the composition includes at least 2, 3, 4, 5, 5, 8 or 10 different siRNA molecules. For example, the composition can include PTK2, EZH2 and Jagged1 siRNAs.
Treating Ovarian Cancer by Altering Activity of an Ovarian Endothelial Cell Tumor-Associated Molecule
Methods are provided to inhibit ovarian endothelial cell tumor-associated molecule activity or expression to treat an ovarian tumor. Treatment of tumors by reducing the number of ovarian endothelial cell tumor-associated molecules can include delaying the development of the tumor in a subject (such as preventing metastasis of a tumor). Treatment of a tumor also includes reducing signs or symptoms associated with the presence of such a tumor (for example by reducing the size, growth or volume of the tumor or a metastasis thereof). Such reduced growth can in some examples decrease or slow metastasis of the tumor, or reduce the size or volume of the tumor by at least 10%, at least 20%, at least 50%, or at least 75%, such as by inhibiting angiogenesis by at least 10%, at least 20%, at least 50%, or at least 75%. For example, ovarian endothelial cell tumor-associated molecules involved in angiogenesis, such as molecules involved in promoting cell proliferation, cell motility or tube formation can be inhibited to treat an ovarian tumor, such as those provided in any of Tables 1, 2, 4 or 5 that are upregulated in ovarian endothelial tumor cells. In other examples, ovarian tumor growth is reduced or inhibited by inhibiting the expression or biological activity ovarian endothelial cell tumor-associated molecules provided in any of Tables 1, 2, 4 or 5 that are upregulated in ovarian tumor endothelial cells. In further examples, inhibition of ovarian endothelial cell tumor-associated molecules includes reducing the invasive activity of the tumor in the subject. In some examples, treatment using the methods disclosed herein prolongs the time of survival of the subject.
Specific Binding Agents
Specific binding agents are agents that selectively bind with higher affinity to a molecule of interest, than to other molecules. For example, a specific binding agent can be one that binds with high affinity to one of the genes or gene products of the ovarian endothelial cell tumor-associated molecules listed in any of Tables 1, 2, 4 or 5 that are upregulated in ovarian tumor endothelial cells, but does not substantially bind to another gene or gene product. In a specific example, a specific binding agent binds to one gene listed in Tables 1, 2, 4 or 5 that is upregulated in ovarian tumor endothelial cells thereby reducing or inhibiting expression of the gene, but does not bind to the other genes (or gene product) listed in such Tables under similar conditions. For example, the agent can interfere with gene expression (transcription, processing, translation, post-translational modification), such as, by interfering with the gene's mRNA and blocking translation of the gene product or by post-translational modification of a gene product, or by causing changes in intracellular localization. In another specific example, a specific binding agent binds to a protein encoded by of one of the genes listed in Table 1, 2, 4 or 5 that is upregulated in ovarian tumor endothelial cells with a binding affinity in the range of 0.1 to 20 nM and reduces or inhibits the activity of such protein.
Examples of specific binding agents include, but are not limited to, siRNAs, antibodies, ligands, recombinant proteins, peptide mimetics, and soluble receptor fragments. One example of a specific binding agent is a siRNA. Methods of making siRNA that can be used clinically are known in the art. Particular siRNAs and methods that can be used to produce and administer them are described in detail below. In some examples, the siRNA is incorporated into a chitosan (CH) nanoparticle, such as chitosan obtained from shellfish or fungi.
Another specific example of a specific binding agent is an antibody, such as a monoclonal or polyclonal antibody. Methods of making antibodies that can be used clinically are known in the art. Particular antibodies and methods that can be used to produce them are described in detail below.
In a further example, small molecular weight inhibitors or antagonists of the receptor protein can be used to regulate activity such as the expression or production of ovarian endothelial cell tumor-associated molecules. In a particular example, small molecular weight inhibitors or antagonists of the proteins encoded by the genes listed in Tables 2 and/or 4 are employed.
Specific binding agents can be therapeutic, for example by reducing or inhibiting the biological activity of a nucleic acid or protein. Complete inhibition is not required. For example, a reduction by at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, or even at least 90% can be sufficient. For example, a specific binding agent that binds with high affinity to a gene listed in Tables 1, 2, 4 and/or 5 that are upregulated in ovarian tumor endothelial cells, may substantially reduce the biological function of the gene or gene product (for example, the ability of the gene or gene product to facilitate angiogenesis). In other examples, a specific binding agent that binds with high affinity to one of the proteins encoded by the genes listed in Tables 1, 2, 4 and/or 5 that are upregulated in ovarian tumor endothelial cells, may substantially reduce the biological function of the protein (for example, the ability of the protein to promote angiogenesis). Such agents can be administered in therapeutically effective amounts to subjects in need thereof, such as a subject having ovarian cancer, such as papillary serous ovarian cancer.
Pre-Screening Subjects
In some examples, subjects are initially screened to determine if they have ovarian cancer. In an example, subjects are initially screened for ovarian cancer by using one of the disclosed gene expression profiles (as discussed in detail below). In some examples, if one or more of the disclosed endothelial cell tumor-associated molecules upregulated in ovarian endothelial cells (such as those listed in Tables 2 and 4) is detected, a specific binding agent capable of reducing or inhibiting ovarian cancer is adminstered.
Pre-Screening Specific Binding Agents
In some examples, specific binding agents are initially screened for treating ovarian cancer by use of the disclosed gene expression profile (see below). For example, the disclosed gene expression profile can be used to identify specific binding agents capable of reducing or inhibiting ovarian cancer. In an example, the disclosed gene expression profile is used to identify compositions that can be employed to reduce or inhibit angiogenesis in ovarian tumors.
Exemplary Tumors
A tumor is an abnormal growth of tissue that results from excessive cell division. A particular example of a tumor is cancer. For example, the current application provides methods for the treatment (such as the prevention or reduction of metastasis) of tumors (such as cancers) by altering the expression/production of one or more disclosed ovarian endothelial cell tumor-associated molecules. In some examples, the tumor is treated in vivo, for example in a mammalian subject, such as a human subject. Exemplary tumors that can be treated using the disclosed methods include, but are not limited to ovarian cancer, including metastases of such tumors to other organs. Generally, the tumor is an ovarian cancer, such as papillary serous ovarian cancer.
Administration
Methods of administrating the disclosed compositions are routine, and can be determined by a skilled clinician. For example, the disclosed therapies (such as those that include a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules listed in Tables 1, 2, 4 or 5 whose expression is increased in ovarian endothelial tumor-associated cells) can be administered via injection, intratumorally, orally, topically, transdermally, parenterally, or via inhalation or spray. In a particular example, a composition is administered intravenously to a mammalian subject, such as a human.
The therapeutically effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In one example, the method includes daily administration of at least 1 μg of the composition to the subject (such as a human subject). For example, a human can be administered at least 1 μg or at least 1 mg of the composition daily, such as 10 μg to 100 μg daily, 100 μg to 1000 μg daily, for example 10 μg daily, 100 μg daily, or 1000 μg daily. In one example, the subject is administered at least 1 μg (such as 1-100 μg) intravenously of the composition including a binding agent that specifically binds to one of the disclosed ovarian endothelial cell tumor-associated molecules provided herein. In one example, the subject is administered at least 1 mg intramuscularly (for example in an extremity) of such composition. In a specific example, the dose is 50 to 350 μg/kg twice weekly, such as 150 μg/kg twice weekly (for example via iv injection). The dosage can be administered in divided doses (such as 2, 3, or 4 divided doses per day), or in a single dosage daily.
In particular examples, the subject is administered the therapeutic composition that includes a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years. In one example, the subject is administered the therapeutic composition that a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules daily for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
The therapeutic compositions, such as those that include a binding agent specific for one of the ovarian endothelial cell tumor-associated molecules, can further include one or more biologically active or inactive compounds (or both), such as anti-neoplastic agents and conventional non-toxic pharmaceutically acceptable carriers, respectively.
In a particular example, a therapeutic composition that includes a therapeutically effective amount of a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules further includes one or more biologically inactive compounds. Examples of such biologically inactive compounds include, but are not limited to: carriers, thickeners, diluents, buffers, preservatives, and carriers. The pharmaceutically acceptable carriers useful for these formulations are conventional (see Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995)). For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can include minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Additional Treatments
In particular examples, prior to, during, or following administration of a therapeutic amount of an agent that reduces or inhibits ovarian cancer due to the interaction of a binding agent with one of the disclosed ovarian endothelial cell tumor-associated molecules, the subject can receive one or more other therapies. In one example, the subject receives one or more treatments to remove or reduce the tumor prior to administration of a therapeutic amount of a composition including a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules.
Examples of such therapies include, but are not limited to, surgical treatment for removal or reduction of the tumor (such as surgical resection, cryotherapy, or chemoembolization), as well as anti-tumor pharmaceutical treatments which can include radiotherapeutic agents, anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents. Particular examples of additional therapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.
“Microtubule binding agent” refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division. Examples of microtubule binding agents that can be used in conjunction with the disclosed therapy include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used and are known to those of ordinary skill in the art. For example, suitable epothilones and epothilone analogs are described in International Publication No. WO 2004/018478. Taxoids, such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.
The following classes of compounds are of use in the methods disclosed herein: Suitable DNA and/or RNA transcription regulators, including, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof also are suitable for use in combination with the disclosed therapies. DNA intercalators and cross-linking agents that can be administered to a subject include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof. DNA synthesis inhibitors suitable for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5′-deoxyuridine, 5-fluorouracil and analogs thereof. Examples of suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof. Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof. Kinase inhibitors include Gleevac, Iressa, and Tarceva that prevent phosphorylation and activation of growth factors.
Other therapeutic agents, for example anti-tumor agents, that may or may not fall under one or more of the classifications above, also are suitable for administration in combination with the disclosed therapies. By way of example, such agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.
In one example, the therapeutic composition (such as one including a binding agent specific for one or more of the disclosed ovarian endothelial cell tumor-associated molecules) is injected into the subject in the presence of an adjuvant. An adjuvant is an agent that when used in combination with an immunogenic agent augments or otherwise alters or modifies a resultant immune response. In some examples, an adjuvant increases the titer of antibodies induced in a subject by the immunogenic agent. In one example, the one or more peptides are administered to the subject as an emulsion with an adjuvant and sterile water for injection (for example an intravenous or intramuscular injection). Incomplete Freund's Adjuvant (Seppic, Inc.) can be used as the Freund's Incomplete Adjuvant (IFA) (Fairfield, N.J.). In some examples, IFA is provided in 3 ml of a mineral oil solution based on mannide oleate (Montanide ISA-51). At the time of injection, the peptide(s) is mixed with the Montanide ISA.51 and then administered to the subject. Other adjuvants can be used, for example, Freund's complete adjuvant, B30-MDP, LA-15-PH, montanide, saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein, hemocyanin, a mycobacterial antigen, and combinations thereof.
In some examples, the subject receiving the therapeutic peptide composition (such as one including a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules) is also administered interleukin-2 (IL-2), for example via intravenous administration. In particular examples, IL-2 (Chiron Corp., Emeryville, Calif.) is administered at a dose of at least 500,000 IU/kg as an intravenous bolus over a 15 minute period every eight hours beginning on the day after administration of the peptides and continuing for up to 5 days. Doses can be skipped depending on subject tolerance.
In some examples, the disclosed compositions can be co-administered with a fully human antibody to cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4). In some example subjects receive at least 1 mg/kg anti-CTLA-4 (such as 3 mg/kg every 3 weeks or 3 mg/kg as the initial dose with subsequent doses reduced to 1 mg/kg every 3 weeks).
In one example, at least a portion of the ovarian tumor (such as a metastatic tumor) is surgically removed (for example via cryotherapy), irradiated, chemically treated (for example via chemoembolization) or combinations thereof, prior to administration of the disclosed therapies (such as administration of a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules). For example, a subject having a metastatic tumor can have all or part of the tumor surgically excised prior to administration of the disclosed therapies (such as one including a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules). In an example, one or more chemotherapeutic agents is administered following treatment with a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules. In another particular example, the subject has a metastatic tumor and is administered radiation therapy, chemoembolization therapy, or both concurrently with the administration of the disclosed therapies (such as one including a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules).
Generation and Administration of siRNA
In an example, certain inhibitors provided by this disclosure are species of siRNAs. One of ordinary skill in the art can readily generate siRNAs which specifically bind to one of the disclosed ovarian endothelial cell tumor-associated molecules that are upregulated in ovarian endothelial cell tumor cells. In an example, commercially available kits, such as siRNA molecule synthesizing kits from PROMEGA® (Madison, Wis.) or AMBION® (Austin, Tex.) may be used to synthesize siRNA molecules. In another example, siRNAs are obtained from commercial sources, such as from QIAGEN® Inc (Germantown, Md.), INVITROGEN® (Carlsbad, Calif.), AMBION (Austin, Tex.), DHARMACON® (Lafayette, Colo.) or OPENBIOSYSTEMS® (Huntsville, Ala.).
In certain examples, expression vectors are employed to express the at least one siRNA molecule. For example, an expression vector can include a nucleic acid sequence encoding at least one siRNA molecule corresponding to at least one of the disclosed ovarian endothelial cell tumor-associated molecules listed in Tables 1, 2, 4 and/or Table 5 that are upregulated in ovarian endothelial cell tumor cells. For example, siRNA specific for EZH2 can be generated using publicly available EZH2 nucleic acid sequences, such as those described above. In a particular example, the vector contains a sequence(s) encoding both strands of a siRNA molecule comprising a duplex. In another example, the vector also contains sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siRNA molecule. Non-limiting examples of such expression vectors are described in Paul et al., Nature Biotechnology 19:505, 2002; Miyagishi and Taira, Nature Biotechnology 19:497, 2002; Lee et al., Nature Biotechnology 19:500, 2002; and Novina et al., Nature Medicine, online publication Jun. 3, 2003.
In other examples, siRNA molecules include a delivery vehicle, including inter alia liposomes, for administration to a subject, carriers and diluents and their salts, and can be present in pharmaceutical compositions. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other delivery vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (see, for example, O'Hare and Normand, International PCT Publication No. WO 00/53722). In one specific example, siRNAs are administered at according to the teachings of Soutschek et al. (Nature Vol. 432: 173-178, 2004) or Karpilow et al. (Pharma Genomics 32-40, 2004) both of which are herein incorporated by reference in their entireties.
In some examples, siRNAs are incorporated into neutral liposomes, such as DOPC or chitosan, and injected intraperitoneal or intravenously. For example, a siRNA can be administered at least 1 μg/kg twice weekly, such as at least 50 μg/kg twice weekly, at least 100 μg/kg twice weekly, at least 125 μg/kg twice weekly, at least 150 μg/kg twice weekly, at least 200 μg/kg twice weekly for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 12 weeks, or at least 24 weeks. In one example, about at least 1-500 μg/kg, such 10-250 μg/kg, is adminstered at least twice weekly for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 12 weeks, or at least 24 weeks. In a certain example, approximately 150 μg/kg is administered twice weekly, for 2 to 3 weeks. In other examples, approximately 1 ug/kg daily for 3 weeks or 50 ug/kg every other day for 3 weeks is administered.
Alternatively, the nucleic acid/vehicle combination can be locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the disclosure, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described by Barry et al., International PCT Publication No. WO 99/31262. Other delivery routes, but are not limited to, oral delivery (such as in tablet or pill form), intrathecal or intraperitoneal delivery. For example, intraperitoneal delivery can take place by injecting the treatment into the peritoneal cavity of the subject in order to directly deliver the molecules to the tumor site. More detailed descriptions of nucleic acid delivery and administration are provided in Sullivan et al., PCT WO 94/02595, Draper et al., PCT WO93/23569, Beigelman et al., PCT WO99/05094, and Klimuk et al., PCT WO99/04819, all of which are incorporated by reference herein.
Alternatively, certain siRNA molecules can be expressed within cells from eukaryotic promoters. Those skilled in the art will recognize that any nucleic acid can be expressed in eukaryotic cells using the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by an enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595).
In other examples, siRNA molecules can be expressed from transcription units (see for example, Couture et al., 1996, TIG 12:510) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. siRNA expressing viral vectors can be constructed based on, for example, but not limited to, adeno-associated virus, retrovirus, adenovirus, lentivirus or alphavirus. In another example, pol III based constructs are used to express nucleic acid molecules (see for example, Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886).
The recombinant vectors capable of expressing the siRNA molecules can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the siRNA molecule interacts with the target mRNA and generates an RNAi response. Delivery of siRNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.
Generation of Antibodies
One of ordinary skill in the art can readily generate antibodies which specifically bind to the disclosed ovarian endothelial cell tumor-associated molecules. These antibodies can be monoclonal or polyclonal. They can be chimeric or humanized. Any functional fragment or derivative of an antibody can be used including Fab, Fab′, Fab2, Fab′2, and single chain variable regions. So long as the fragment or derivative retains specificity of binding for the ovarian endothelial cell tumor-associated molecule it can be used in the methods provided herein. Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to appropriate antigen at least 2, at least 5, at least 7 or 10 times more than to irrelevant antigen or antigen mixture, then it is considered to be specific.
In an example, monoclonal antibodies are generated to the ovarian endothelial cell tumor-associated molecules disclosed in Tables 1, 2, 4 or 5 that are upregulated in ovarian endothelial cell tumor cells. These monoclonal antibodies each include a variable heavy (VH) and a variable light (VL) chain and specifically bind to the specific ovarian endothelial cell tumor-associated molecules. For example, the antibody can bind the specific ovarian endothelial cell tumor-associated molecules with an affinity constant of at least 106 M−1, such as at least 107 M−1, at least 108 M−1, at least 5×108 M−1, or at least 109 M−1.
The specific antibodies can include a VL polypeptide having amino acid sequences of the complementarity determining regions (CDRs) that are at least about 90% identical, such as at least about 95%, at least about 98%, or at least about 99% identical to the amino acid sequences of the specific ovarian endothelial cell tumor-associated molecules and a VH polypeptide having amino acid sequences of the CDRs that are at least about 90% identical, such as at least about 95%, at least about 98%, or at least about 99% identical to the amino acid sequences of the specific ovarian endothelial cell tumor-associated molecules.
In one example, the sequence of the specificity determining regions of each CDR is determined Residues that are outside the SDR (non-ligand contacting sites) are substituted. For example, in any of the CDR sequences, at most one, two or three amino acids can be substituted. The production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well known in the art. For example, humanized antibodies can be routinely produced. The antibody or antibody fragment can be a humanized immunoglobulin having CDRs from a donor monoclonal antibody that binds one of the disclosed ovarian endothelial cell tumor-associated molecules and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks. Generally, the humanized immunoglobulin specifically binds to one of the disclosed ovarian endothelial cell tumor-associated molecules with an affinity constant of at least 107 M−1, such as at least 108 M−1 at least 5×108 M−1 or at least 109 M−1.
In another example, human monoclonal antibodies to the disclosed ovarian endothelial cell tumor-associated molecules in Tables 1, 2, 4 and 5 that are upregulated in ovarian endothelial tumor cells are produced. Human monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from heavy and light variable chains of the donor mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. For example, when mouse monoclonal antibodies are used therapeutically, the development of human anti-mouse antibodies (HAMA) leads to clearance of the murine monoclonal antibodies and other possible adverse events. Chimeric monoclonal antibodies, with human constant regions, humanized monoclonal antibodies, retaining only murine CDRs, and “fully human” monoclonal antibodies made from phage libraries or transgenic mice have all been used to reduce or eliminate the murine content of therapeutic monoclonal antibodies.
Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993. The antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgG1, IgG2, IgG3 and IgG4.
In one example, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Human framework regions, and mutations that can be made in a humanized antibody framework regions, are known in the art (see, for example, in U.S. Pat. No. 5,585,089).
Antibodies, such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab′)2, and Fv, which include a heavy chain and light chain variable region and are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with their epitope. These fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′)2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988. Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain. To produce these antibodies, the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. If the VH and the VL are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
In an additional example, the Fv fragments include VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271, 1993; and Sandhu, supra).
Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
One of skill will realize that conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pI and low toxicity of the molecules Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the VH and the VL regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
In some examples, naked antibodies can be administered at least 5 mg per kg every two weeks, such as at least 10 mg per kg, at least 25 mg per kg, at least 30 mg per kg, at least 50 mg per kg (for example, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg per kg), at least once a week, at least once every 2 weeks, at least once every 3 weeks, or at least once every month depending upon the ovarian cancer. In an example, the antibodies are administered continuously. In another example, antibodies or antibody fragments conjugated to cytotoxic agents (immunotoxins) are administered at least 10 μg per kg, such as at least 20, at least 30, at least 50, at least 70, at least 100 μg per kg, at least twice a week, at least once a week, at least once every two weeks, at least once every month depending upon the ovarian cancer. In one example, 50 μg per kg is administered twice a week for 2 to 3 weeks. In other examples, the subject is administered the therapeutic composition that a binding agent specific for one or more of the disclosed ovarian endothelial cell tumor-associated molecules daily for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months. Subjects can be monitored by methods known to those skilled in the art to determine ovarian tumor responsiveness to the antibody treatment. The subject can be monitored by non invasive techniques such as CT or MRI imaging to assess tumor response. It is contemplated that additional agents can be administered, such as antineoplastic agents in combination with or following treatment with the antibodies.
Methods of Evaluating the Effectiveness of an Ovarian Tumor Treatment
Methods are disclosed herein for determining the effectiveness of a binding agent specific for one of the disclosed ovarian endothelial cell tumor-associated molecules) for the treatment of an ovarian tumor in a subject with the ovarian tumor. In an example, the method includes detecting expression of an ovarian endothelial cell tumor-associated molecule in a sample from the subject following administration of the binding agent (such as an siRNA), for example at least 24 hours, at least 1 week, at least 2 weeks, or at least 4 weeks following administration of the agent. The expression of the ovarian endothelial cell tumor-associated molecule following administration can be compared to a control, such as a reference value. An alteration in the expression of the ovarian endothelial cell tumor-associated molecule relative to the control following administration indicates that the agent is effective for the treatment of the ovarian cancer in the subject.
In a specific example, the method includes detecting and comparing the protein expression levels of the ovarian endothelial cell tumor-associated molecules. In other examples, the method includes detecting and comparing the mRNA expression levels of the ovarian endothelial cell tumor-associated molecules. In certain examples, the treatment is considered effective if the expression levels are altered by at least 2-fold, such as by at least 3-fold, at least 4-fold, at least 6-fold or at least 10-fold relative to the control.
In one example, the specific ovarian endothelial cell tumor-associated molecule is detected in a biological sample. In a particular example, the biological sample is a tumor biopsy. In another example, the ovarian endothelial cell tumor-associated molecule is detected in a serum sample. For example, the ovarian endothelial cell tumor-associated molecule is detected in a serum sample if the specific molecule is known to be secreted or located on a cell surface susceptible to enzymatic cleavage.
Altering Ovarian Endothelial Cell Tumor-Associated Molecules' Activity Such as Expression
In an example, an alteration in the expression of one or more of the disclosed ovarian endothelial cell tumor-associated molecules following administration includes an increase or decrease in production of a gene product/expression, such as RNA or protein. For example, an alteration can include processes that downregulate or decrease transcription of a gene or translation of mRNA whose expression or activity is increased in ovarian endothelial tumor cells. Gene downregulation includes any detectable decrease in the production of a gene product. In certain examples, production/expression of a gene product decreases by at least 2-fold, for example at least 3-fold, at least 4-fold, at least 6-fold, or at least 10-fold as compared to a control. Exemplary ovarian endothelial cell tumor-associated molecules that are up-regulated in ovarian tumor endothelial cells are presented in Tables 1, 2, 4 and 5. Thus, a decrease in the expression of one or more of the molecules listed in Tables 1, 2, 4 or 5 that is noted as being up-regulated in ovarian tumor endothelial cells following treatment indicates that the agent is of use for treating the ovarian cancer.
Exemplary ovarian endothelial cell tumor-associated molecules that are down-regulated in ovarian tumor endothelial cells are presented in Table 1 with specific examples provided in Table 3. Thus, an increase in the expression of one or more of the molecules listed in Table 3 following administration indicates that the agent is effective for the treatment of ovarian cancer.
In another example, an alteration can include processes that increase transcription of a gene or translation of mRNA. Gene up-regulation includes any detectable increase in the production of a gene product. In certain examples, production/expression of a gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, at least 6-fold, at least 10-fold or at least 28-fold following treatment as compared to a control.
Detection of Ovarian Endothelial Cell Tumor-Associated Nucleic Acids
Nucleic acids can be detected by any method known in the art. In some examples, nucleic acids are isolated, amplified, or both, prior to detection. In an example, the biological sample can be incubated with primers that permit the amplification of one or more of the disclosed ovarian endothelial cell tumor-associated mRNAs, under conditions sufficient to permit amplification of such products. For example, the biological sample is incubated with probes that can bind to one or more of the disclosed ovarian endothelial cell tumor-associated nucleic acid sequences (such as cDNA, genomic DNA, or RNA (such as mRNA)) under high stringency conditions. The resulting hybridization can then be detected using methods known in the art. In one example, the effectiveness of an ovarian tumor treatment is identified by applying isolated nucleic acid molecules to an array in which the isolated nucleic acid molecules are obtained from a biological sample including ovarian endothelial cancer cells following treatment with the ovarian tumor treatment. In such example, the array includes oligonucleotides complementary to all ovarian endothelial cell tumor-associated genes listed in Table 1. In a particular example, the array is a commercially available array such as a U133 Plus 2.0 oligonucleotide array from AFFYMETRIX® (AFFYMETRIX®, Santa Clara, Calif.).
In an example, the isolated nucleic acid molecules are incubated with the array including oligonucleotides complementary to the ovarian endothelial cell tumor-associated molecules that are up-regulated in ovarian tumor endothelial cells, such as those listed in Table 2, Table 3, Table 4 and/or Table 5 for a time sufficient to allow hybridization between the isolated nucleic acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecule:oligonucleotide complexes. The isolated nucleic acid molecule:oligonucleotide complexes are then analyzed to determine if expression of the isolated nucleic acid molecules is altered. In such example, an ovarian tumor treatment is effective if a decrease in the expression of ovarian endothelial tumor-associated molecules is observed as compared to a control (such as a normal endothelial cell) or reference value. In an additional example, the array includes oligonucleotides complementary to the ovarian endothelial cell tumor-associated molecules that down-regulated in ovarian tumor endothelial cells, such as those listed in Table 3. In this example, an ovarian tumor treatment is effective if an increase in the expression of one or more ovarian endothelial tumor-associated molecules is observed.
Gene Expression Profile
A gene expression profile is disclosed herein that can be used to identify the effectiveness of an ovarian tumor treatment. In an example, the gene expression profile includes at least two of the ovarian endothelial cell tumor-associated molecules listed in Table 1, such as at least 5, at least 7, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, or at least 1100 molecules (for example, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 175, 225, 275, 325, 350, 375, 450, 550, 650, 750, 850, 950, 1050 or 1149 of those listed).
In a particular example, the gene expression profile includes at least 2, at least 5, at least 7, at least 10, at least 20, at least 25, at least 27 molecules (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28 or 29 molecules) listed in Table 2, Table 4 and/or Table 5 that are associated with an at least six-fold increase in expression in tumor endothelial cells. In a particular example, the at least two molecules include EGFL6 and TNFAIP6. In other particular examples, the at least two ovarian endothelial cell tumor-associated molecules include EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, and PLXDC1.
In other particular examples, the gene expression profile includes at least 2, at least 5, at least 7, at least 10, at least 13, or at least 15 molecules (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 molecules) that are down-regulated in ovarian tumor endothelial cells as listed in Table 3. For example, the profile includes the seventeen ovarian endothelial cell tumor-associated molecules listed in Table 3.
Detecting Ovarian Endothelial Cell Tumor-Associated Proteins
As an alternative to analyzing the sample for the presence of nucleic acids, the presence of proteins can be determined. Proteins can be detected by any method known in the art. In some examples, proteins are purified prior to detection. For example, the effect of an ovarian tumor treatment can be determined by incubating the biological sample with one or more antibodies that specifically binds to one of the disclosed ovarian endothelial cell tumor-associated proteins encoded by the genes listed in Tables 1, Table 2, Table 3, Table 4 or Table 5 to detect expression. The primary antibody can include a detectable label. For example, the primary antibody can be directly labeled, or the sample can be subsequently incubated with a secondary antibody that is labeled (for example with a fluorescent label). The label can then be detected, for example by microscopy, ELISA, flow cytometry, or spectrophotometry. In another example, the biological sample is analyzed by Western blotting for the presence or absence of the specific ovarian endothelial cell tumor-associated molecule. In other examples, the biological sample is analyzed by mass spectrometry for the presence or absence of the specific ovarian endothelial cell tumor-associated molecule.
In one example, the antibody that specifically binds an ovarian endothelial cell tumor-associated molecule (such as those listed in Table 1) is directly labeled with a detectable label. In another example, each antibody that specifically binds an ovarian endothelial cell tumor-associated molecule (the first antibody) is unlabeled and a second antibody or other molecule that can bind the human antibody that specifically binds the respective ovarian endothelial cell tumor-associated molecule is labeled. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody can be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125I, 131I, 35S or 3H.
In an alternative example, ovarian endothelial cell tumor-associated molecules can be assayed in a biological sample by a competition immunoassay utilizing ovarian endothelial cell tumor-associated molecule standards labeled with a detectable substance and unlabeled antibody that specifically bind to the desired ovarian endothelial cell tumor-associated molecule. In this assay, the biological sample (such as serum, tissue biopsy, or cells isolated from a tissue biopsy), the labeled ovarian endothelial cell tumor-associated molecule standards and the antibody that specifically binds to ovarian endothelial cell tumor-associated molecule are combined and the amount of labeled ovarian endothelial cell tumor-associated molecule standard bound to the unlabeled antibody is determined. The amount of ovarian endothelial cell tumor-associated molecule in the biological sample is inversely proportional to the amount of labeled ovarian endothelial cell tumor-associated molecule standard bound to the antibody that specifically binds the ovarian endothelial cell tumor-associated molecule.
Identifying Agents to Treat Ovarian Cancer
Methods are provided herein for identifying agents to treat an ovarian cancer. For example, agents that decrease expression or activity of a gene that is upregulated in ovarian endothelial tumor cells (such as those listed in Tables 2 and 4), as well as agents that increase activity of a gene that is downregulated in ovarian endothelial tumor cells (such as those listed in Table 3), can be identified using these methods. In an example, the method includes contacting an ovarian tumor endothelial cell with one or more test agents under conditions sufficient for the one or more test agents to alter the activity of at least one ovarian endothelial cell tumor-associated molecule listed in any of Tables 1-5. It is contemplated that several doses of the agent can be tested and then expression levels of nucleic acids or proteins can be determined. The method also includes detecting the activity or expression of the at least one ovarian endothelial cell tumor-associated molecule in the presence and absence of the one or more test agents. The activity or expression of the at least one ovarian endothelial cell tumor-associated molecule in the presence of the one or more test agents is then compared to the activity or expression of the at least one ovarian endothelial cell tumor-associated molecule in the absence of such agents to determine if there is differential expression of the at least one ovarian endothelial cell tumor associated molecule. In several examples, differential expression of the ovarian endothelial cell tumor-associated molecule in the presence of the agent (as compared to expression in the absence of the agent) indicates that the one or more test agents is of use to treat the ovarian tumor.
In an example, determining whether there is differential expression of one or more ovarian endothelial cell tumor-associated molecules includes generating a gene expression profile for the subject. For example, a gene expression profile for the subject can be generated by using an array of molecules including an ovarian endothelial cell tumor-associated expression profile.
Ovarian Endothelial Cell Tumor-Associated Molecules
Ovarian endothelial cell tumor-associated molecules can include nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins. In a specific example, detecting differential expression of the ovarian endothelial cell tumor-associated molecules includes detecting differential mRNA expression of the disclosed ovarian endothelial cell tumor-associated molecules. For example, such differential expression can be measured by real time quantitative polymerase chain reaction or microarray analysis or other methods known in the art. In another example, detecting differential expression of the ovarian endothelial cell tumor-associated molecules includes detecting differential protein expression of the disclosed ovarian endothelial cell tumor-associated molecules. For example, protein differential expression is measured by Western blot analysis or a protein microarray.
Test Agents
The one or more test agents can be any substance, including, but not limited to, a protein (such as an antibody), a nucleic acid molecule (such as a siRNA), an organic compound, an inorganic compound, a small molecule or any other molecule of interest. In a particular example, the test agent is a siRNA that reduces or inhibits the activity (such as the expression) of one of the ovarian endothelial cell tumor-associated molecules listed in Tables 2, 4 or 5. For example, the siRNA is directed to an ovarian endothelial cell tumor-associated molecule listed in Table 2, 4 or 5 which is involved in angiogenesis, such as a molecule that is involved in at least one of cell proliferation, tube formation or cell motility.
In other examples, the test agent is an antibody. For example, the antibody is directed to specifically bind to an ovarian endothelial cell tumor-associated protein encoded by one of the genes listed in any of Tables 1, 2, 4 or 5 that are upregulated in ovarian endothelial tumor cells. In a particular example, the antibody is directed to an ovarian endothelial cell tumor-associated protein encoded by one of the genes listed in Tables 2, 4 or 5 that is upregulated in ovarian endothelial tumor cells and which is involved in angiogenesis, such as a gene that is involved in at least one of cell proliferation, tube formation or cell motility. In another example, the test agent is a nucleic acid encoding one or more of the proteins listed in Table 3. For example, the nucleic acid can be part of a vector suitable for gene therapy.
Altering Ovarian Endothelial Cell Tumor-Associated Molecules' Activity
In an example, an alteration in the activity of one or more of the disclosed ovarian endothelial cell tumor-associated molecules includes an increase or decrease in production of a gene product, such as RNA or protein. For example, an alteration can include processes that downregulate or decrease transcription of a gene or translation of mRNA. Gene downregulation includes any detectable decrease in the production of a gene product. In certain examples, production/expression of a gene product decreases by at least 2-fold, for example at least 3-fold, at least 4-fold, at least 6-fold, or at least 10-fold as compared to a control (such as a reference value or a normal endothelial cell). For example, a decrease in one or more of the disclosed ovarian endothelial cell tumor-associated molecules up-regulated in ovarian tumor endothelial cells (such as those listed in Tables 2, 4 and 5), is indicative of an agent that is effective at treating ovarian cancer.
In another example, an alteration can include processes that increase transcription of a gene or translation of mRNA. Gene up-regulation includes any detectable increase in the production of a gene product. In certain examples, production/expression of a gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, at least 6-fold, at least 10-fold or at least 28-fold as compared to a control. For example, an increase in one or more of the disclosed ovarian endothelial cell tumor-associated molecules down-regulated in ovarian tumor endothelial cells (such as those listed in Table 3) is indicative of an agent that is effective at treating ovarian cancer.
Detection of Ovarian Endothelial Cell Tumor-Associated Nucleic Acids
Nucleic acids can be detected by any method known in the art, such as those described above. In one example, a therapeutic agent is identified by applying isolated nucleic acid molecules to an array in which the isolated nucleic acid molecules are obtained from a biological sample including ovarian endothelial cancer cells following treatment with the one or more test agents. In such example, the array includes oligonucleotides complementary to all ovarian endothelial cell tumor-associated genes listed in Table 1. In a particular example, the array is a commercially available array such as a U133 Plus 2.0 oligonucleotide array from AFFYMETRIX® (AFFYMETRIX®, Santa Clara, Calif.).
In an example, the isolated nucleic acid molecules are incubated with the array including oligonucleotides complementary to the ovarian endothelial cell tumor-associated molecules listed in Table 2, 4 and/or 5 for a time sufficient to allow hybridization between the isolated nucleic acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecule:oligonucleotide complexes. The isolated nucleic acid molecule:oligonucleotide complexes are then analyzed to determine if expression of the isolated nucleic acid molecules is altered. In such example, an agent is considered is effective if the test agent decreases expression of ovarian endothelial tumor-associated molecules upregulated in ovarian endothelial cell tumors relative to the absence of the agent (such as a decrease of at least 2-, 3-, 4-, 5- or 10-fold). Similarly, an agent is considered is effective if the test agent increases expression of ovarian endothelial tumor-associated molecules downregulated in ovarian endothelial cell tumors relative to the absence of the agent (such as an increase of at least 2-, 3-, 4-, 5- or 10-fold).
Gene Expression Profile
The disclosed gene profile (as described above) can also be used to identify agents to treat an ovarian tumor, such as a cancer, in a subject. In an example, the gene expression profile includes at least two of the ovarian endothelial cell tumor-associated molecules listed in Table 1, such as at least 5, at least 7, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, or at least 1100 molecules (for example, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 175, 225, 275, 325, 350, 375, 450, 550, 650, 750, 850, 950, 1050 or 1149 of those listed).
In a particular example, the gene expression profile includes at least 2, at least 5, at least 7, at least 10, at least 20, at least 25, or at least 27 molecules (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28 or 29 molecules) listed in Table 2, 4 and/or 5 that are associated with an at least six-fold increase in expression in tumor endothelial cells. In a particular example, the at least two molecules include EGFL6 and TNFAIP6. In other particular examples, the at least two ovarian endothelial cell tumor-associated molecules include EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, and PLXDC1.
In other particular examples, the gene expression profile includes at least 2, at least 5, at least 7, at least 10, at least 13, or at least 15 molecules (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 molecules) that are down-regulated in ovarian tumor endothelial cells as listed in Table 3. For example, the profile includes the seventeen ovarian endothelial cell tumor-associated molecules listed in Table 3.
Detecting Ovarian Endothelial Cell Tumor-Associated Proteins
As an alternative to analyzing the sample for the presence of nucleic acids, the presence of proteins can be determined using any method known in the art. In some examples, proteins are purified before detection. For example, the effect of one or more test agents on an ovarian tumor can be determined by incubating the biological sample with an antibody that specifically binds to one of the disclosed ovarian endothelial cell tumor-associated proteins encoded by the genes listed in Tables 1-5. The primary antibody can include a detectable label. For example, the primary antibody can be directly labeled, or the sample can be subsequently incubated with a secondary antibody that is labeled (for example with a fluorescent label). The label can then be detected, for example by microscopy, ELISA, flow cytometry, or spectrophotometry. In another example, the biological sample is analyzed by Western blotting for the presence or absence of the specific ovarian endothelial cell tumor-associated molecule. In some examples, the biological sample is analyzed by mass spectrometry.
In one example, the antibody that specifically binds an ovarian endothelial cell tumor-associated molecule (such as those listed in Table 1) is directly labeled with a detectable label. In another example, each antibody that specifically binds an ovarian endothelial cell tumor-associated molecule (the first antibody) is unlabeled and a second antibody or other molecule that can bind the human antibody that specifically binds the respective ovarian endothelial cell tumor-associated molecule is labeled. As is well known to one of skill in the art, a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody can be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125I, 131I, 35S or 3H.
In an alternative example, ovarian endothelial cell tumor-associated molecules can be assayed in a biological sample by a competition immunoassay utilizing ovarian endothelial cell tumor-associated molecule standards labeled with a detectable substance and an unlabeled antibody that specifically binds the desired ovarian endothelial cell tumor-associated molecule. In this assay, the biological sample (such as serum, tissue biopsy, or cells isolated from a tissue biopsy), the labeled ovarian endothelial cell tumor-associated molecule standards and the antibody that specifically binds the desired ovarian endothelial cell tumor-associated molecule are combined and the amount of labeled ovarian endothelial cell tumor-associated molecule standard bound to the unlabeled antibody is determined. The amount of ovarian endothelial cell tumor-associated molecule in the biological sample is inversely proportional to the amount of labeled ovarian endothelial cell tumor-associated molecule standard bound to the antibody that specifically binds the ovarian endothelial cell tumor-associated molecule.
Methods of Diagnosing and Prognosing an Ovarian Tumor
Metastasis is a major complication in the pathogenesis of tumors, such as ovarian cancer, and is typically indicative of poor prognosis. It is also known that angiogenesis is a crucial factor in the progression of solid tumors and metastases, including ovarian cancer. The formation of the vascular stroma plays an important role in the pathophysiology of malignancy. For instance, in the absence of vascular support tumors may become necrotic, or even apoptotic. In contrast, the onset of angiogenesis marks a phase of rapid proliferation, local invasion, and ultimately metastasis.
Without wishing to be bound to a particular theory, it is proposed that an alteration in the expression of the disclosed ovarian endothelial tumor-associated molecules associated with angiogenesis, such as molecules involved in cell proliferation, cell motility or tube formation, including those disclosed in FIG. 5 (such as EZH2), is related to enhanced ovarian tumor cell metastasis and a poor clinical outcome. Thus, methods of diagnosing or prognosing an ovarian tumor that overexpresses at least one pro-angiogenic ovarian endothelial cell tumor-associated molecule (such as those listed in Tables 1, 2, 4 and 5 that are upregulated in ovarian endothelial tumor cells; e.g., EZH2) or underexpresses at least one proangiogenic ovarian endothelial cell tumor associated molecule (such as those listed in Tables 1 and 3 that are downregulated in ovarian endothelial tumor cells), are disclosed. In some examples, such methods can be used to identify those subjects that will benefit from the disclosed treatment methods. For example, such diagnostic methods can be performed prior to the subject undergoing the treatments described above. In other examples, these methods are utilized to predict the metastatic potential of the ovarian cancer, a poor prognosis, or combinations thereof. In one particular example, these methods are utilized to predict a poor prognosis, such as to indicate a decreased survival time.
In an example, the method includes detecting expression of at least one angiogenic ovarian endothelial cell tumor-associated molecule listed in Tables 1-5, such as at least two, at least 5, at least 7, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200 ovarian endothelial cell tumor-associated molecules related to angiogenesis (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, or 250 listed in Tables 1-5) in a sample from the subject exhibiting one or more symptoms associated with ovarian cancer.
In an example, the specific angiogenic ovarian endothelial cell tumor-associated molecule, such as EZH2, is detected in a biological sample. In a particular example, the biological sample is a sample taken from a subject with ovarian epithelial cancer. In a particular example, the biological sample is a tumor biopsy. In another example, the angiogenic ovarian endothelial cell tumor-associated molecule is detected in a serum sample. For example, the ovarian endothelial cell tumor-associated molecule is detected in a serum sample if the specific molecule is secreted or located on a cell surface susceptible to enzymatic cleavage.
In one example, detection of at least one angiogenic ovarian endothelial cell tumor-associated molecule listed in any of Tables 1, 2, 3, 4 or 5, such as detection of EZH2, in a biological sample from the subject is used to diagnose or prognose an ovarian tumor. Methods of detecting such molecules in a sample are known in the art and are routine. In some examples, the relative amount of pro-angiogenic ovarian endothelial cell tumor-associated molecules present is determined, for example by quantitating the expression level of such molecules. For example, the relative or absolute quantity of the at least one angiogenic ovarian endothelial cell tumor-associated molecule in a sample can be determined.
The activity such as the expression level of the disclosed pro-angiogenic ovarian endothelial cell tumor-associated molecules in a sample obtained from a subject is compared to a control. In one example, an increase in expression of one or more of the angiogenic ovarian endothelial cell tumor-associated molecules upregulated in ovarian tumor endothelial cells (such as those listed in Table 2) as compared to a non-tumor control or reference value indicates the presence of an ovarian tumor, the ovarian tumor is metastatic, the ovarian tumor is likely to become metastatic, a poor prognosis or a combination thereof. In some examples, a decrease in expression of one or more of the angiogenic ovarian endothelial cell tumor-associated molecules that is downregulated in ovarian tumor endothelial cells (such as those listed in Table 3 or VASH1) as compared to a non-tumor control or reference value indicates the presence of an ovarian tumor, the ovarian tumor is metastatic, the ovarian tumor is likely to become metastatic, a poor prognosis or a combination thereof.
For example, the level of the angiogenic ovarian endothelial cell tumor-associated molecules, such as the level of EZH2, detected can be compared to a non-tumor control or reference value, such as a value that represents a level of angiogenic ovarian endothelial cell tumor-associated molecules expected if an ovarian tumor is or is not metastatic or is a low grade tumor or early stage tumor. In one example, the angiogenic ovarian endothelial cell tumor-associated molecules detected in a tumor sample are compared to the level of such molecules detected in a sample obtained from a subject that does not have an ovarian tumor or has a non-metastatic ovarian tumor. In certain examples, detection of at least a 2-fold, such as by at least 3-fold, at least 4-fold, at least 6-fold or at least 10-fold alteration in the relative amount of the pro-angiogenic ovarian endothelial cell tumor-associated molecules in a tumor sample, as compared to the relative amount of such molecules in a control indicates that the subject has a tumor with metastatic potential, has a tumor that has metastasized, has a poor prognosis, or combinations thereof. In some examples, detection of statistically similar relative amounts of pro-angiogenic ovarian endothelial cell tumor-associated molecules observed in a tumor sample, as compared to the relative amount of such molecules in a control sample that is not metastatic, indicates that that subject does not have a tumor with metastatic potential, does not have a tumor that has metastasized, has a good prognosis, or combinations thereof.
In a specific example, the method includes detecting and comparing the nucleic acid expression levels of the pro-angiogenic ovarian endothelial cell tumor-associated molecules such as DNA, cDNA, or mRNAs. In a specific example, the method includes detecting and comparing the mRNA expression levels of the pro-angiogenic ovarian endothelial cell tumor-associated molecules. For example, such expression can be measured by real time quantitative polymerase chain reaction or microarray analysis. In a particular example, the disclosed gene expression profile is utilized to diagnosis and/or prognosis an ovarian tumor.
Detection of Ovarian Endothelial Cell Tumor-Associated Nucleic Acids
Nucleic acids can be detected by any method known in the art. In some examples, nucleic acids are isolated, amplified, or both, prior to detection. In an example, the biological sample can be incubated with primers that permit the amplification of one or more of the disclosed ovarian endothelial cell tumor-associated mRNAs, under conditions sufficient to permit amplification of such products. For example, the biological sample is incubated with probes that can bind to one or more of the disclosed ovarian endothelial cell tumor-associated nucleic acid sequences (such as cDNA, genomic DNA, or RNA (such as mRNA)) under high stringency conditions. The resulting hybridization can then be detected using methods known in the art. In one example, a therapeutic agent is identified by applying isolated nucleic acid molecules to an array in which the isolated nucleic acid molecules are obtained from a biological sample including ovarian endothelial cancer cells for example following treatment with the one or more test agents. In such example, the array includes oligonucleotides complementary to all ovarian endothelial cell tumor-associated genes listed in Table 1. In a particular example, the array is a commercially available array such as a U133 Plus 2.0 oligonucleotide array from AFFYMETRIX® (AFFYMETRIX®, Santa Clara, Calif.).
In an example, the isolated nucleic acid molecules are incubated with the array including oligonucleotides complementary to the ovarian endothelial cell tumor-associated molecules listed in Tables 2, 3, 4 and/or 5 for a time sufficient to allow hybridization between the isolated nucleic acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecule:oligonucleotide complexes. In a particular example, the isolated nucleic acid molecules are incubated with the array including oligonucleotides complementary to at least EZH2. The isolated nucleic acid molecule:oligonucleotide complexes are then analyzed to determine if expression of the isolated nucleic acid molecules is altered.
Gene Expression Profile
The disclosed gene profile (as described above) can also be used in the diagnosis and prognosis of an ovarian tumor in a subject. In an example, the gene expression profile includes at least two of the ovarian endothelial cell tumor-associated molecules listed in Table 1, such as at least 5, at least 7, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, or at least 1100 molecules (for example, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 175, 225, 275, 325, 350, 375, 450, 550, 650, 750, 850, 950, 1050 or 1149 of those listed).
In a particular example, the gene expression profile includes at least 1, at least 3, at least 5, at least 7, at least 10, at least 20, at least 25, or at least 27 molecules (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28 or 29 molecules) listed in Table 2, 4 and/or 5 that are associated with angiogenesis, such as molecules involved in cell proliferation, cell motility and/or tube formation. In a particular example, the at least one molecule includes EZH2
In other particular examples, the gene expression profile includes at least 2, at least 5, at least 7, at least 10, at least 13, or at least 15 molecules (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 molecules) that are down-regulated in ovarian tumor endothelial cells as listed in Table 3. For example, the profile includes the seventeen ovarian endothelial cell tumor-associated molecules listed in Table 3.
Detecting Ovarian Endothelial Cell Tumor-Associated Proteins
As an alternative to analyzing the sample for the presence of nucleic acids, alterations in protein expression can be measured by methods known in the art such as Western blot analysis, mass spectrometry, immunoassay or a protein microarray (as described above). For example, the metastatic potential of an ovarian tumor can be determined by using a protein array that includes one or more capture agents, such as antibodies that are specific for the one or more disclosed ovarian endothelial tumor-associated molecules that are related to angiogenesis, such as molecules that play a role in cell proliferation, cell motility or tube formation, such as EZH2.
The disclosure is further illustrated by the following non-limiting Examples.
Example 1
Materials and Methods for Examples 2-7
Sample Preparation.
Fresh tissue samples (5 normal ovaries and 10 epithelial high-grade, stage III or IV invasive serous ovarian cancers) were obtained from patients undergoing primary surgical exploration at the M.D. Anderson Cancer Center. The minced tissue was digested with collagenase A, elastase and DNase 1 at 37° C. for 90 minutes to yield a single cell suspension. A number of negative selections followed including removal of platelets and red blood cells (RBCs) by Percoll separation, removal of epithelial cells using M450 beads, which are prebound to BerEP4 antibody, removal of leukocytes using anti-CD-14, CD-45, and CD-64 beads (Dynal Biotech, Brown Deer, Wis.). Positive selection was performed with P1H12 (CD 146) immunobeads (P1H12 antibody was from Chemicon, Temecula, Calif.), and the beads linked to secondary antibody were from Dynal Biotech. Immunostaining was then performed using von Willebrand factor and 4′,6-diamidino-2-phenylindole nuclear staining to confirm the purification of endothelial cells.

Total RNA Amplification for AFFYMETRIX® GENECHIP® Hybridization and Image Acquisition.

To successfully generate sufficient labeled cRNA for microarray analysis from 25 ng of total RNA, two rounds of amplification were necessary. For the first round synthesis of double-stranded cDNA, 25 ng of total RNA was reverse transcribed using the Two-Cycle cDNA Synthesis Kit (AFFYMETRIX®, Santa Clara, Calif.) and oligo-dT24-T7








(SEQ ID NO: 1: 5′-GGCCAGTGAATTGTAATACGACTCACTATAGG


GAGGCGG-3′)






primer according to the manufacturer's instructions followed by amplification with the MEGA script T7 Kit (Ambion, Inc., Austin, Tex.). After cleanup of the cRNA with a GENECHIP® Sample Cleanup Module IVT column (AFFYMETRIX®), second round double stranded cDNA was amplified using the IVT Labeling Kit (AFFYMETRIX®). A 15.0 μg aliquot of labeled product was fragmented by heat and ion-mediated hydrolysis at 94° C. for 35 minutes in 24 μl H2O and 6 μl of 5× Fragmentation Buffer (AFFYMETRIX®). The fragmented cRNA was hybridized for 16 hr at 45° C. in a Hybridization Oven 640 to a U133 plus 2.0 oligonucleotide array (AFFYMETRIX®). Washing and staining of the arrays with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, Oreg.) was completed in a Fluidics Station 450 (AFFYMETRIX®). The arrays were then scanned using a confocal laser GENECHIP® Scanner 3000 and GENECHIP® Operating Software (AFFYMETRIX®).

Data Normalization and Filtering.

Global normalization at a target value of 500 was applied to all 15 of the arrays under consideration using GENECHIP® Operating Software (AFFYMETRIX®). Normalized data were uploaded into the National Cancer Institute's Microarray Analysis Database (mAdb) for quality control screening and collation prior to downstream analyses. Biometric Research Branch (BRB) ArrayTools version 3.2.2 software developed by Drs. Richard Simon and Amy Peng Lam of the Biometrics Research Branch of the National Cancer Institute was used to filter and complete the statistical analysis of the array data. BRB-ArrayTools is a multifunctional Excel add-in that contains utilities for processing and analyzing microarray data using the R version 2.0.1 environment (R Development Core Team, 2004). Of the 47,000 transcripts represented on the array, hybridization control probe sets and probe sets scored as absent at α1=0.05 or marginal (M) at α 2=0.065 were excluded. In addition, only those transcripts present in greater than 50% of the arrays and displaying a variance in the top 50th percentile were evaluated.

Class Comparison Analysis.

Differentially expressed genes were identified for tumor and normal endothelial cell specimens using a multivariate permutation test in BRB-ArrayTools (Simon et al., “Design and Analysis of DNA Microarray Investigations” Springer-Verlag, 2003). A total of 2000 permutations were completed to identify the list of probe sets with a false discovery rate less than 10% at a confidence of 95%. Differential expression was considered significant at a p<0.001. A random-variance t-test was selected to permit the sharing of information among probe sets within class variation without assuming that all of the probe sets possess the same variance (Wright et al., Bioinformatics 19: 2448-2455, 2003). A global assessment of whether expression profiles were different between classes was also performed. During each permutation the class labels were reassigned randomly and the p-value for each probe set recalculated. The proportion of permutations yielding at least as many significant genes as the actual data set at a p-value<0.001 was reported as the significance level of the global test.

Pathway Analysis.

Differentially regulated genes identified in a series of 48 late-stage (III and IV) high-grade (Hurwitz et al., N. Engl. J. Med. 350: 2335-2342, 2004) microdissected papillary serous ovarian carcinomas, as compared to 10 normal ovarian surface epithelial brushings (Bonome et al., Cancer Res. 65: 10602-10612, 2005), were categorized by cellular component according to the Gene Ontology (GO) ontological hierarchy. Epithelial genes associated with the cell membrane, extracellular matrix, and extracellular region were used as central nodes to identify signaling pathways modulated in tumor-associated endothelial cell isolates. This was accomplished using PathwayAssist version 3.0 software (Iobion Informatics LLC, La Jolla, Calif.). This software package contains over 500,000 documented protein interactions acquired from MedLine using the natural language processing algorithm MEDSCAN. The proprietary database can be used to develop a biological association network (BAN) to identify putative co-regulated signaling pathways using expression data.

qRT-PCR Validation.

Quantitative real-time PCR (qRT-PCR) was performed on 100 ng of double-amplified product from the 15 specimens using primer sets specific for 23 select genes, and the housekeeping genes GAPDH, GUSB, and cyclophilin. An iCycler iQ Real-Time PCR Detection System (BIORAD® Laboratories, Hercules, Calif.) was used in conjunction with the QuantiTect SYBR Green RT-PCR Kit (QIAGEN® Inc., Valencia, Calif.) according to previously described cycling conditions (Donninger et al., Oncogene 23: 8065-8077, 2004). To calculate the relative expression for each gene, the 2-ΔΔCT method was used averaging the CT values for the three housekeeping genes for a single reference gene value (Livak and Schmittgen, Methods 25: 402-408, 2001).

Immunohistochemical Staining.

Paraffin sections were stained for the following antibodies: rabbit anti-Fyn at 1:400 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or rabbit anti-Fak at 1:50, mouse anti-MMP-9 at 1:40 (Oncogene-Research Products, Boston, Mass.), anti-β2-Arrestin at 1:200 (Santa Cruz Biotechnology, Inc.), anti-PLXDC1 at 1:200 (Abcam, Inc., Cambridge, Mass.), or anti-Jagged1 at 1:200 (Santa Cruz Biotechnology, Inc.) diluted in PBS at 4° C. After three washes in PBS, sections were incubated with secondary antibody for 1 hr at room temperature. Positive reactions were rendered visible by incubating the slides with stable 3,3-diaminobenzidine for 5-10 min. The sections were rinsed with distilled water, counterstained with Gill's hematoxylin for 30 s, and mounted with Universal Mount (Research Genetics, Huntsville, Ala.). The intensity of protein expression in the endothelial cells was evaluated using OPTIMAS 6.5 software and the mean optical density (OD) was calculated from 5 normal ovarian and 5 ovarian cancer samples. Ten vessels were selected randomly from each sample for the measurements.

Small interfering RNA (siRNA).

The small interfering RNA (siRNA) constructs were purchased from QIAGEN® (Germantown, Md.): a control sequence with no homology to any human mRNA (as determined by BLAST search), and separate sequences designed to target EZH2, Jagged1, or PTK2 mRNA. The Jagged1 siRNA target sequence is SEQ ID NO: 2 (5′-CTGCATTTAGGGAGTATTCTA-3′). The EZH2 siRNA was targeted to the region corresponding to residues 85-106 of human EZH2 (Gene accession No. NM004456; 5′-AACCATGTTTACAACTATCAA-3; SEQ ID NO: 3). The EZH2 siRNA sense sequence was 5′-CCAUGUUUACAACUAUCAAtt-3; SEQ ID NO: 4) and the antisense sequence was 3′-ttGGUACAAAUGUUGAUAGUU-5; SEQ ID NO: 5). For in vitro delivery, siRNA (5 μg) was incubated with 30 μL RNAiFect transfection reagent (QIAGEN®) for 10 min at room temperature and added to cells in culture at 80% confluence in 35 mm culture plates.

Cell Migration Assay.

Unstimulated motility was determined in membrane invasion culture system chambers containing polycarbonate filter (with 10 μm pores) that had been soaked in 0.1% gelatin, as described previously (Sood et al., Am. J. Pathol. 165: 1087-1095, 2004). HUVECs (1×105) were seeded in each upper well, allowed to incubate at 37° C. for 6 hr in Dulbecco's modified Eagle's medium (DMEM) containing 15% serum, and subsequently processed as described for the invasion assay.

Tube Formation Assay.

Matrigel (12.5 mg/ml) was thawed at 4° C. and 50 μl were quickly added to each well of a 96-well plate and allowed to solidify for 10 min at 37° C. The wells were then incubated for 6 h at 37° C. with HUVECs (20,000 cells/well), which had previously been treated for 18 h with the indicated siRNA. The formation of capillary-like structures was examined microscopically and photographs (50×) were taken using a RETIGA® 1300 camera and a ZEISS® Axiovert S100 microscope. The extent to which capillary-like structures formed in the gel was quantified by analysis of digitized images to determine the thread length of the capillary-like network, using a commercially available image analysis program (Northern Eclipse, North Tonawanda, N.Y.).

Example 2
Purity of Isolated Endothelial Cells
This example illustrates the purity of the endothelial cell samples utilized in the disclosed microarray analyses.
According to the methods described in Example 1, samples were immunostained with endothelial cell markers P1H12 and von Willebrand factor to determine endothelial cell purity. Immunostaining revealed that the employed purification technique yielded endothelial cell purity of >95% in all samples. Thus, the disclosed isolation technique resulted in a highly pure population of endothelial cells.
Example 3
Development of Gene Expression Profile for Ovarian Tumor-Endothelial Cells
This example provides a gene expression profile for ovarian tumor endothelial cells.
According to the methods described in Example 1, gene expression differences in purified endothelial cells from 10 invasive epithelial ovarian cancers and 5 normal (non-tumor) ovaries were determined by using the AFFYMETRIX® Human U133 Plus 2.0 Gene Chip platform. The nucleic acid sequence of each AFFYMETRIX® probe listed in the tables below is herein incorporated by reference, and is available from the AFFYMETRIX®website. As illustrated in Table 1, 1149 genes were identified as being differentially regulated ≧2-fold in endothelium derived from epithelial ovarian cancers compared to normal ovarian tissue. A positive fold change indicates the gene was upregulated in ovarian endothelial tumor sample and a negative fold change indicates the gene was downregulated in such sample. A multivariate permutation t-test (p<0.001) providing 95% confidence that the number of false discoveries did not exceed 10% of the complete gene list identified. In addition, global analysis of the gene list returned a p value<5×104.







TABLE 1







Gene expression profile.












Affymetrix
Parametric
Fold Change





Probe Set
P-Value
(Tumor/Normal)
Gene Symbol
Description
Map















117_at
0.0005215
4.4
HSPA6
heat shock 70 kDa protein 6
1q23






(HSP70B′) (HSPA6), mRNA.


1552365_at
0.0008711
6.6
SCIN
scinderin (SCIN), mRNA.
7p21.3


1552767_a_at
0.0002277
−6.5
HS6ST2
heparan sulfate 6-O-
Xq26.2






sulfotransferase 2 (HS6ST2),






mRNA.


1552790_a_at
0.0009635
−2.2
TLOC1
translocation protein 1
3q26.2






(TLOC1), mRNA.


1552889_a_at
0.0003238
2.5
XTP7
protein 7 transactivated by
19q13.32






hepatitis B virus X antigen






(HBxAg) (XTP7), mRNA.


1553185_at
0.000128
4.6
RASEF
RAS and EF-hand domain
9q21.32






containing


1553186_x_at
3.33E−05
5.3
RASEF
RAS and EF-hand domain
9q21.32






containing


1553407_at
0.0003912
2.5
MACF1
Glycine-rich protein (GRP3S)
1p32-p31


1553538_s_at
0.000594
2.2

Unknown


1553569_at
0.0001391
2.6

Unknown


1553570_x_at
0.0002337
2.5

Unknown


1553575_at
2.80E−06
4.6

Unknown


1553909_x_at
0.0005681
3.8
C10orf6
Chromosome 10 open reading
10q24.32






frame 6


1553959_a_at
0.0009778
2.3
B3GALT6
UDP-Gal:betaGal beta 1,3-
1p36.33






galactosyltransferase






polypeptide 6 (B3GALT6),






mRNA.


1553983_at
0.000152
2.5
DTYMK
deoxythymidylate kinase
2q37.3






(thymidylate kinase) (DTYMK),






mRNA.


1554168_a_at
0.0003894
2.1
SH3KBP1
SH3-domain kinase binding
Xp22.1-p21.3






protein 1 (SH3KBP1), transcript






variant 2, mRNA.


1554309_at
0.0006114
2.3
EIF4G3
Eukaryotic translation initiation
1p36.12






factor 4 gamma, 3


1554334_a_at
7.15E−05
5.2
DNAJA4
DnaJ (Hsp40) homolog,
15q25.1






subfamily A, member 4






(DNAJA4), mRNA.


1554455_at
0.0007696
2.4
LINS1
lines homolog 1 (Drosophila)
15q26.3






(LINS1), transcript variant 2,






mRNA.


1554464_a_at
0.000263
−2.8
CRTAP
cartilage associated protein
3p22.3






(CRTAP), mRNA.


1554595_at
0.0005225
2.6
SYMPK
Symplekin
19q13.3


1554640_at
0.0004537
2.6
PALM2-AKAP2
Paralemmin 2
9q31-q33


1554678_s_at
0.0002435
−2
HNRPDL
Heterogeneous nuclear
4q13-q21






ribonucleoprotein D-like


1554703_at
0.0008778
2.1
ARHGEF10
Rho guanine nucleotide
8p23






exchange factor (GEF) 10


1555014_x_at
0.0004322
3.5

OK/SW-cl.92


1555241_at
0.0001337
3.2

Hypothetical gene supported by
8q21.2






BC055092


1555243_x_at
0.0001753
2.8

Hypothetical gene supported by
8q21.2






BC055092


1555374_at
0.0003108
4
TTL
Tubulin tyrosine ligase
2q13


1555823_at
0.0001833
−2

BS 3076
14


1556126_s_at
1.04E−05
3.5
GPATC2
G patch domain containing 2
1q41


1556138_a_at
0.0001589
2.9
COL5A1
Collagen, type V, alpha 1
9q34.2-q34.3


1556185_a_at
0.0002463
2.6

CDNA clone IMAGE: 5260162
7


1556242_a_at
3.82E−05
2.1

Homo sapiens, clone
8






IMAGE: 3885623, mRNA


1556316_s_at
0.0005561
3.3
MIF
Macrophage migration
22q11.23






inhibitory factor (glycosylation-






inhibiting factor)


1556499_s_at
0.0005246
2.5
COL1A1
collagen, type I, alpha 1
17q21.3-q22.1






(COL1A1), mRNA.


1556835_s_at
0.0002018
2.2

Transcribed locus
11


1557432_at
0.0001321
3.6
RASAL2
RAS protein activator like 2
1q24


1557527_at
0.0003572
2.5
RUNX1
Runt-related transcription factor
21q22.3






1 (acute myeloid leukemia 1;






aml1 oncogene)


1558019_at
2.09E−05
−2.9
DST
Dystonin
6p12-p11


1558048_x_at
2.01E−05
4.1

Unknown


1558292_s_at
0.0007105
2
PIGW
phosphatidylinositol glycan,
17q12






class W (PIGW), mRNA.


1558426_x_at
0.000189
2.9

Chromosome 7 open reading
7






frame 19


1558487_a_at
0.0002657
−2.6
TMED4
Transmembrane emp24 protein
7p13






transport domain containing 4


1558836_at
5.35E−05
3.2

MRNA; cDNA
2






DKFZp667A182 (from clone






DKFZp667A182)


1559060_a_at
5.16E−05
3
KIAA1961
KIAA1961 gene
5q23.3


1559078_at
6.58E−05
4.6
BCL11A
B-cell CLL/lymphoma 11A
2p16.1






(zinc finger protein)


1559101_at
9.12E−05
2.6
FYN
FYN oncogene related to SRC,
6q21






FGR, YES


1559410_at
4.66E−05
3.6

Unknown


1559436_x_at
4.54E−05
4.8
ARRB2
Arrestin, beta 2
17p13


1559585_at
0.000489
3.2
FLJ31033
Hypothetical protein FLJ31033
4q32.3


1559593_a_at
0.0005238
2.9
CRSP7
Cofactor required for Sp1
19p13.11






transcriptional activation,






subunit 7, 70 kDa


1560817_at
0.0001564
2.5
MOV10
Mov10, Moloney leukemia virus
1p13.2






10, homolog (mouse)


1562062_at
0.000114
4

Homo sapiens transcribed






sequence with weak similarity to






protein ref: NP_055301.1






(H. sapiens) neuronal thread






protein [Homo sapiens]


1562063_x_at
0.0003481
3.4

Homo sapiens transcribed






sequence with weak similarity to






protein ref: NP_055301.1






(H. sapiens) neuronal thread






protein [Homo sapiens]


1562270_at
0.000382
4
ARHGEF7
Rho guanine nucleotide
13q34






exchange factor (GEF) 7


1562271_x_at
1.81E−05
3.9
ARHGEF7
Rho guanine nucleotide
13q34






exchange factor (GEF) 7


1562456_at
0.0008617
2.3

MRNA; cDNA
11






DKFZp566C0924 (from clone






DKFZp566C0924)


1563357_at
0.0002718
3.4
SERPINB9
serpin peptidase inhibitor, clade
6p25






B (ovalbumin), member 9






(SERPINB9), mRNA.


1565579_at
0.0003108
3.7
TATDN2
TatD DNase domain containing 2
3p25.3


1565823_at
1.91E−05
4.8
7-Sep
septin 7 (SEPT7), transcript
7p14.3-p14.1






variant 2, mRNA.


1565974_at
0.0008723
2.8
SUV420H1
Suppressor of variegation 4-20
11q13.2






homolog 1 (Drosophila)


1566887_x_at
8.32E−05
4.3
KIAA0284
KIAA0284
14q32.33


1568619_s_at
0.0002665
2.2
LOC162073
Hypothetical protein
16p12.3






LOC162073


1568954_s_at
0.0001751
2.2

Unknown


1569519_at
0.0006199
2.5
FLJ21272
hypothetical protein FLJ21272
1q21.2


1569872_a_at
0.0004892
2.4

Homo sapiens, clone
16






IMAGE: 5242623


1570061_at
8.83E−05
3.2

CDNA clone IMAGE: 4555030
3


1570143_at
0.0005727
2.4

Homo sapiens, clone
8






IMAGE: 3932570, mRNA


1570185_at
0.0003081
3

Homo sapiens, clone
10






IMAGE: 5766850, mRNA


200005_at
0.0005509
−2
EIF3S7
eukaryotic translation initiation
22q13.1






factor 3, subunit 7 zeta,






66/67 kDa (EIF3S7), mRNA.


200010_at
0.0001997
−2.2
RPL11
Ribosomal protein L11
1p36.1-p35


200013_at
2.50E−05
−2.6
RPL24
ribosomal protein L24 (RPL24),
3q12






mRNA.


200021_at
0.0001035
2.5
CFL1
cofilin 1 (non-muscle) (CFL1),
11q13






mRNA.


200022_at
0.0002003
−2.1
RPL18
ribosomal protein L18 (RPL18),
19q13






mRNA.


200023_s_at
1.61E−05
−2.4
EIF3S5
eukaryotic translation initiation
11p15.4






factor 3, subunit 5 epsilon,






47 kDa (EIF3S5), mRNA.


200024_at
0.0004964
−2.5
RPS5
ribosomal protein S5 (RPS5),
19q13.4






mRNA.


200074_s_at
0.0001721
−2.2
RPL14
ribosomal protein L14 (RPL14),
3p22-p21.2






mRNA.


200081_s_at
6.20E−06
−3.3
RPS6
ribosomal protein S6 (RPS6),
9p21






mRNA.


200642_at
0.0007047
−2
SOD1
superoxide dismutase 1, soluble
21q22.11






(amyotrophic lateral sclerosis 1






(adult)) (SOD1), mRNA.


200651_at
0.0001597
−2.3
GNB2L1
guanine nucleotide binding
5q35.3






protein (G protein), beta






polypeptide 2-like 1 (GNB2L1),






mRNA.


200665_s_at
0.00039
2.7
SPARC
secreted protein, acidic,
5q31.3-q32






cysteine-rich (osteonectin)






(SPARC), mRNA.


200676_s_at
0.0007037
−2.5
UBE2L3
ubiquitin-conjugating enzyme
22q11.21






E2L 3 (UBE2L3), transcript






variant 1, mRNA.


200700_s_at
4.15E−05
2.3
KDELR2
KDEL (Lys-Asp-Glu-Leu)
7p22.1






endoplasmic reticulum protein






retention receptor 2 (KDELR2),






mRNA.


200734_s_at
6.61E−05
2.5
ARF3
ADP-ribosylation factor 3
12q13






(ARF3), mRNA.


200735_x_at
9.63E−05
−2.1
NACA
nascent-polypeptide-associated
12q23-q24.1






complex alpha polypeptide






(NACA), mRNA.


200755_s_at
0.0007055
2.1
CALU
calumenin (CALU), mRNA.
7q32


200760_s_at
5.53E−05
−2.6
ARL6IP5
ADP-ribosylation-like factor 6
3p14






interacting protein 5 (ARL6IP5),






mRNA.


200806_s_at
0.0008155
−2
HSPD1
heat shock 60 kDa protein 1
2q33.1






(chaperonin) (HSPD1), nuclear






gene encoding mitochondrial






protein, transcript variant 2,






mRNA.


200811_at
1.25E−05
−3.6
CIRBP
cold inducible RNA binding
19p13.3






protein (CIRBP), mRNA.


200827_at
0.0004906
2.3
PLOD1
procollagen-lysine 1,2-
1p36.3-p36.2






oxoglutarate 5-dioxygenase 1






(PLOD1), mRNA.


200866_s_at
0.0004451
−2.1
PSAP
prosaposin (variant Gaucher
10q21-q22






disease and variant






metachromatic leukodystrophy)






(PSAP), mRNA.


200883_at
7.19E−05
−3.2
UQCRC2
ubiquinol-cytochrome c
16p12






reductase core protein II






(UQCRC2), mRNA.


200906_s_at
0.0002674
−3.2
KIAA0992
palladin (KIAA0992), mRNA.
4q32.3


200920_s_at
0.0001341
−2.3
BTG1
B-cell translocation gene 1, anti-
12q22






proliferative (BTG1), mRNA.


200937_s_at
5.83E−05
−2.4
RPL5
ribosomal protein L5 (RPL5),
1p22.1






mRNA.


200951_s_at
0.000314
4.1
CCND2
cyclin D2 (CCND2), mRNA.
12p13


200953_s_at
0.0001997
2.6
CCND2
cyclin D2 (CCND2), mRNA.
12p13


200965_s_at
0.000112
−5.9
ABLIM1
actin binding LIM protein 1
10q25






(ABLIM1), transcript variant 4,






mRNA.


200999_s_at
0.0009198
2
CKAP4
cytoskeleton-associated protein
12q23.3






4 (CKAP4), mRNA.


201008_s_at
0.0009045
−2.7
TXNIP
thioredoxin interacting protein
1q21.1






(TXNIP), mRNA.


201009_s_at
0.0004645
−2.8
TXNIP
thioredoxin interacting protein
1q21.1






(TXNIP), mRNA.


201018_at
0.0007096
−2.3
EIF1AX
eukaryotic translation initiation
Xp22.12






factor 1A, X-linked (EIF1AX),






mRNA.


201023_at
2.69E−05
−2.4
TAF7
TAF7 RNA polymerase II,
5q31






TATA box binding protein






(TBP)-associated factor, 55 kDa






(TAF7), mRNA.


201030_x_at
2.40E−05
−2.2
LDHB
lactate dehydrogenase B
12p12.2-p12.1






(LDHB), mRNA.


201036_s_at
0.0005734
−3.7
HADHSC
L-3-hydroxyacyl-Coenzyme A
4q22-q26






dehydrogenase, short chain






(HADHSC), mRNA.


201054_at
1.41E−05
−2.2
HNRPA0
heterogeneous nuclear
5q31






ribonucleoprotein A0






(HNRPA0), mRNA.


201076_at
1.82E−05
−2.4
NHP2L1
NHP2 non-histone chromosome
22q13.2-q13.31






protein 2-like 1 (S. cerevisiae)






(NHP2L1), transcript variant 2,






mRNA.


201085_s_at
0.000222
−2.3
SON
SON DNA binding protein
21q22.11






(SON), transcript variant a,






mRNA.


201088_at
9.10E−06
2.7
KPNA2
karyopherin alpha 2 (RAG
17q23.1-q23.3






cohort 1, importin alpha 1)






(KPNA2), mRNA.


201101_s_at
3.64E−05
−2.9
BCLAF1
BCL2-associated transcription
6q22-q23






factor 1 (BCLAF1), mRNA.


201129_at
0.0002614
−2.6
SFRS7
splicing factor, arginine/serine-
2p22.1






rich 7, 35 kDa (SFRS7),






transcript variant 1, mRNA.


201133_s_at
0.0001686
−2.5
PJA2
praja 2, RING-H2 motif
2p22.1






containing (PJA2), mRNA.


201154_x_at
0.0001159
−2.2
RPL4
ribosomal protein L4 (RPL4),
15q22






mRNA.


201163_s_at
0.0003882
2.2
IGFBP7
insulin-like growth factor
4q12






binding protein 7 (IGFBP7),






mRNA.


201193_at
0.0004945
−2.9
IDH1
isocitrate dehydrogenase 1
2q33.3






(NADP+), soluble (IDH1),






mRNA.


201204_s_at
1.43E−05
2.2
RRBP1
Ribosome binding protein 1
20p12






homolog 180 kDa (dog)


201206_s_at
0.0002058
2.5
RRBP1
ribosome binding protein 1
20p12






homolog 180 kDa (dog)






(RRBP1), mRNA.


201250_s_at
0.0001573
3.1
SLC2A1
solute carrier family 2
1p35-p31.3






(facilitated glucose transporter),






member 1 (SLC2A1), mRNA.


201261_x_at
0.0003831
4.3
BGN
biglycan (BGN), mRNA.
Xq28


201302_at
1.57E−05
−3.1
ANXA4
annexin A4 (ANXA4), mRNA.
2p13


201370_s_at
0.0002857
−2.3
CUL3
cullin 3 (CUL3), mRNA.
2q36.3


201376_s_at
0.0001535
−2.4
HNRPF
heterogeneous nuclear
10q11.21-q11.22






ribonucleoprotein F (HNRPF),






mRNA.


201408_at
3.95E−05
−3.2
PPP1CB
protein phosphatase 1, catalytic
2p23






subunit, beta isoform (PPP1CB),






transcript variant 2, mRNA.


201425_at
1.80E−06
−2.9
ALDH2
aldehyde dehydrogenase 2
12q24.2






family (mitochondrial)






(ALDH2), nuclear gene






encoding mitochondrial protein,






mRNA.


201427_s_at
0.0004665
−2.5
SEPP1
selenoprotein P, plasma, 1
5q31






(SEPP1), mRNA.


201431_s_at
0.0003957
−4.5
DPYSL3
dihydropyrimidinase-like 3
5q32






(DPYSL3), mRNA.


201432_at
2.09E−05
−3.3
CAT
catalase (CAT), mRNA.
11p13


201455_s_at
0.0004393
−2.7
NPEPPS
aminopeptidase puromycin
17q21






sensitive (NPEPPS), mRNA.


201482_at
0.0004221
−2.9
QSCN6
quiescin Q6 (QSCN6), transcript
1q24






variant 2, mRNA.


201484_at
0.0001619
−2.2
SUPT4H1
suppressor of Ty 4 homolog 1
17q21-q23






(S. cerevisiae) (SUPT4H1),






mRNA.


201487_at
0.0002325
3.1
CTSC
cathepsin C (CTSC), transcript
11q14.1-q14.3






variant 1, mRNA.


201496_x_at
0.0005336
−6.1
MYH11
myosin, heavy polypeptide 11,
16p13.13-p13.12






smooth muscle (MYH11),






transcript variant SM2, mRNA.


201506_at
0.0003278
3.3
TGFBI
transforming growth factor,
5q31






beta-induced, 68 kDa (TGFBI),






mRNA.


201529_s_at
0.0001633
−3
RPA1
replication protein A1, 70 kDa
17p13.3






(RPA1), mRNA.


201535_at
6.72E−05
−2.3
UBL3
ubiquitin-like 3 (UBL3),
13q12-q13






mRNA.


201554_x_at
0.0003257
−2.5
GYG
glycogenin (GYG), mRNA.
3q24-q25.1


201579_at
0.0007798
2.6
FAT
FAT tumor suppressor homolog
4q34-q35






1 (Drosophila) (FAT), mRNA.


201581_at
2.11E−05
−2.4
DJ971N18.2
Hypothetical protein
20p12






DJ971N18.2


201584_s_at
0.0001736
2.3
DDX39
DEAD (Asp-Glu-Ala-Asp) box
19p13.12






polypeptide 39 (DDX39),






transcript variant 1, mRNA.


201596_x_at
1.62E−05
4.3
KRT18
keratin 18 (KRT18), transcript
12q13






variant 2, mRNA.


201600_at
3.48E−05
−2.3
PHB2
prohibitin 2 (PHB2), mRNA.
12p13


201666_at
0.0004812
3.4
TIMP1
TIMP metallopeptidase inhibitor
Xp11.3-p11.23






1 (TIMP1), mRNA.


201674_s_at
0.0001795
−3
AKAP1
A kinase (PRKA) anchor protein
17q21-q23






1 (AKAP1), nuclear gene






encoding mitochondrial protein,






transcript variant 1, mRNA.


201696_at
0.000121
−2
SFRS4
splicing factor, arginine/serine-
1p35.3






rich 4 (SFRS4), mRNA.


201697_s_at
0.0001256
2.4
DNMT1
DNA (cytosine-5-)-
19p13.2






methyltransferase 1 (DNMT1),






mRNA.


201712_s_at
0.0008422
−2.2
RANBP2
RAN binding protein 2
2q12.3






(RANBP2), mRNA.


201737_s_at
5.10E−06
−3.1
6-Mar
membrane-associated ring finger
5p15.2






(C3HC4) 6 (MARCH6), mRNA.


201756_at
0.0003533
−2
RPA2
replication protein A2, 32 kDa
1p35






(RPA2), mRNA.


201810_s_at
0.0001904
−3.2
SH3BP5
SH3-domain binding protein 5
3p24.3






(BTK-associated) (SH3BP5),






transcript variant 2, mRNA.


201816_s_at
0.0004168
−2.2
GBAS
glioblastoma amplified sequence
7p12






(GBAS), mRNA.


201871_s_at
0.0001348
−2
LOC51035
ORF (LOC51035), mRNA.
11q12.3


201891_s_at
0.0007795
2
B2M
beta-2-microglobulin (B2M),
15q21-q22.2






mRNA.


201893_x_at
0.0006272
−3.1
DCN
decorin (DCN), transcript
12q21.33






variant B, mRNA.


201911_s_at
0.0001467
3.4
FARP1
FERM, RhoGEF (ARHGEF)
13q32.2






and pleckstrin domain protein 1






(chondrocyte-derived) (FARP1),






transcript variant 1, mRNA.


201922_at
0.0001538
−2
TINP1
TGF beta-inducible nuclear
5q13.3






protein 1 (TINP1), mRNA.


201960_s_at
0.0002203
−2
MYCBP2
MYC binding protein 2
13q22






(MYCBP2), mRNA.


201973_s_at
0.0004504
2
C7orf28A
chromosome 7 open reading
7p22.1






frame 28A (C7orf28A), mRNA.


202016_at
0.0001808
3.2
MEST
mesoderm specific transcript
7q32






homolog (mouse) (MEST),






transcript variant 3, mRNA.


202028_s_at
0.0002331
3
RPL38
ribosomal protein L38 (RPL38),
17q23-q25






mRNA.


202029_x_at
1.93E−05
−2.1
RPL38
ribosomal protein L38 (RPL38),
17q23-q25






mRNA.


202037_s_at
0.0002558
−3.1
SFRP1
secreted frizzled-related protein
8p12-p11.1






1 (SFRP1), mRNA.


202068_s_at
0.0001171
−4.1
LDLR
low density lipoprotein receptor
19p13.3






(familial hypercholesterolemia)






(LDLR), mRNA.


202073_at
4.49E−05
−2.6
OPTN
optineurin (OPTN), transcript
10p13






variant 2, mRNA.


202105_at
3.00E−07
−3.2
IGBP1
immunoglobulin (CD79A)
Xq13.1-q13.3






binding protein 1 (IGBP1),






mRNA.


202119_s_at
0.0002779
−3.2
CPNE3
copine III (CPNE3), mRNA.
8q21.3


202139_at
0.0004872
−2.1
AKR7A2
aldo-keto reductase family 7,
1p35.1-p36.23






member A2 (aflatoxin aldehyde






reductase) (AKR7A2), mRNA.


202148_s_at
0.0006205
2.2
PYCR1
pyrroline-5-carboxylate
17q25.3






reductase 1 (PYCR1), transcript






variant 2, mRNA.


202156_s_at
0.0004765
−2.8
CUGBP2
CUG triplet repeat, RNA
10p13






binding protein 2 (CUGBP2),






transcript variant 2, mRNA.


202157_s_at
9.00E−07
−6.1
CUGBP2
CUG triplet repeat, RNA
10p13






binding protein 2 (CUGBP2),






transcript variant 2, mRNA.


202158_s_at
2.99E−05
−4.2
CUGBP2
CUG triplet repeat, RNA
10p13






binding protein 2 (CUGBP2),






transcript variant 2, mRNA.


202172_at
0.0006154
−2.4
ZNF161
zinc finger protein 161
17q23.2






(ZNF161), mRNA.


202202_s_at
0.0002996
3.4
LAMA4
laminin, alpha 4 (LAMA4),
6q21






mRNA.


202214_s_at
0.0005849
−2
CUL4B
Cullin 4B
Xq23


202232_s_at
0.0002955
−2.2
hfl-B5
dendritic cell protein (hfl-B5),
11p13






mRNA.


202259_s_at
3.74E−05
−3.2
PFAAP5
phosphonoformate immuno-
13q12-q13






associated protein 5 (PFAAP5),






mRNA.


202260_s_at
0.0005909
−2.2
STXBP1
syntaxin binding protein 1
9q34.1






(STXBP1), transcript variant 2,






mRNA.


202286_s_at
0.0003361
5.5
TACSTD2
tumor-associated calcium signal
1p32-p31






transducer 2 (TACSTD2),






mRNA.


202292_x_at
0.0001802
2.4
LYPLA2
lysophospholipase II (LYPLA2),
1p36.12-p35.1






mRNA.


202297_s_at
6.25E−05
2.2
RER1
RER1 retention in endoplasmic
1pter-q24






reticulum 1 homolog (S. cerevisiae)






(RER1), mRNA.


202314_at
0.0001115
−2.6
CYP51A1
cytochrome P450, family 51,
7q21.2-q21.3






subfamily A, polypeptide 1






(CYP51A1), mRNA.


202350_s_at
0.0002121
−3.7
MATN2
matrilin 2 (MATN2), transcript
8q22






variant 2, mRNA.


202364_at
0.0002246
−2.5
MXI1
MAX interactor 1 (MXI1),
10q24-q25






transcript variant 3, mRNA.


202378_s_at
6.45E−05
−2.2
LEPROT
leptin receptor overlapping
1p31.2






transcript (LEPROT), mRNA.


202404_s_at
0.000452
2.9
COL1A2
collagen, type I, alpha 2
7q22.1






(COL1A2), mRNA.


202429_s_at
0.0001255
−2.4
PPP3CA
protein phosphatase 3 (formerly
4q21-q24






2B), catalytic subunit, alpha






isoform (calcineurin A alpha)






(PPP3CA), mRNA.


202464_s_at
0.0009823
3.5
PFKFB3
6-phosphofructo-2-
10p14-p15






kinase/fructose-2,6-






biphosphatase 3


202465_at
9.80E−06
4.8
PCOLCE
procollagen C-endopeptidase
7q22






enhancer (PCOLCE), mRNA.


202468_s_at
7.84E−05
−2.5
CTNNAL1
catenin (cadherin-associated
9q31.2






protein), alpha-like 1






(CTNNAL1), mRNA.


202502_at
5.08E−05
−2
ACADM
acyl-Coenzyme A
1p31






dehydrogenase, C-4 to C-12






straight chain (ACADM),






nuclear gene encoding






mitochondrial protein, mRNA.


202510_s_at
1.14E−05
3.7
TNFAIP2
tumor necrosis factor, alpha-
14q32






induced protein 2 (TNFAIP2),






mRNA.


202512_s_at
6.24E−05
−2.2
APG5L
APG5 autophagy 5-like (S. cerevisiae)
6q21






(APG5L), mRNA.


202536_at
0.0002234
−2
CHMP2B
chromatin modifying protein 2B
3p12.1






(CHMP2B), mRNA.


202546_at
0.0004572
5.6
VAMP8
vesicle-associated membrane
2p12-p11.2






protein 8 (endobrevin)






(VAMP8), mRNA.


202547_s_at
6.98E−05
3
ARHGEF7
Rho guanine nucleotide
13q34






exchange factor (GEF) 7






(ARHGEF7), transcript variant






1, mRNA.


202573_at
0.0002474
2
CSNK1G2
casein kinase 1, gamma 2
19p13.3






(CSNK1G2), mRNA.


202581_at
0.0002675
2.8
HSPA1B
heat shock 70 kDa protein 1B
6p21.3






(HSPA1B), mRNA.


202630_at
6.64E−05
−2.7
APPBP2
amyloid beta precursor protein
17q21-q23






(cytoplasmic tail) binding






protein 2 (APPBP2), mRNA.


202665_s_at
0.0003645
2.9
WASPIP
Wiskott-Aldrich syndrome
2q31.1






protein interacting protein






(WASPIP), mRNA.


202722_s_at
0.0002949
2.1
GFPT1
glutamine-fructose-6-phosphate
2p13






transaminase 1 (GEPT1),






mRNA.


202723_s_at
2.20E−06
−4
FOXO1A
forkhead box O1A
13q14.1






(rhabdomyosarcoma)






(FOXO1A), mRNA.


202724_s_at
6.00E−07
−3.3
FOXO1A
forkhead box O1A
13q14.1






(rhabdomyosarcoma)






(FOXO1A), mRNA.


202731_at
0.0008149
−2.6
PDCD4
programmed cell death 4
10q24






(neoplastic transformation






inhibitor) (PDCD4), transcript






variant 1, mRNA.


202733_at
0.0005751
3
P4HA2
procollagen-proline, 2-
5q31






oxoglutarate 4-dioxygenase






(proline 4-hydroxylase), alpha






polypeptide II (P4HA2),






transcript variant 3, mRNA.


202746_at
0.0005864
−6.4
ITM2A
integral membrane protein 2A
Xq13.3-Xq21.2






(ITM2A), mRNA.


202749_at
0.0009431
−2.2
WRB
Tryptophan rich basic protein
21q22.3


202761_s_at
0.0003908
−2.3
SYNE2
spectrin repeat containing,
14q23.2






nuclear envelope 2 (SYNE2),






transcript variant 4, mRNA.


202780_at
0.0009309
−2
OXCT1
3-oxoacid CoA transferase 1
5p13.1






(OXCT1), nuclear gene






encoding mitochondrial protein,






mRNA.


202820_at
1.90E−05
3.3
AHR
aryl hydrocarbon receptor
7p15






(AHR), mRNA.


202825_at
0.0004002
−2.1
SLC25A4
solute carrier family 25
4q35






(mitochondrial carrier; adenine






nucleotide translocator),






member 4 (SLC25A4), nuclear






gene encoding mitochondrial






protein, mRNA.


202888_s_at
0.0005702
4.7
ANPEP
alanyl (membrane)
15q25-q26






aminopeptidase (aminopeptidase






N, aminopeptidase M,






microsomal aminopeptidase,






CD13, p150) (ANPEP), mRNA.


202899_s_at
5.55E−05
−2.8
SFRS3
splicing factor, arginine/serine-
6p21






rich 3 (SFRS3), mRNA.


202908_at
5.90E−06
−3.4
WFS1
Wolfram syndrome 1
4p16






(wolframin) (WFS1), mRNA.


202911_at
0.0008837
−2.1
MSH6
mutS homolog 6 (E. coli)
2p16






(MSH6), mRNA.


202920_at
4.60E−06
−4.2
ANK2
ankyrin 2, neuronal (ANK2),
4q25-q27






transcript variant 2, mRNA.


202952_s_at
2.37E−05
7.6
ADAM12
ADAM metallopeptidase
10q26.3






domain 12 (meltrin alpha)






(ADAM12), transcript variant 1,






mRNA.


202954_at
0.0006434
2.8
UBE2C
ubiquitin-conjugating enzyme
20q13.12






E2C (UBE2C), transcript variant






1, mRNA.


202957_at
0.000374
2.7
HCLS1
hematopoietic cell-specific Lyn
3q13






substrate 1 (HCLS1), mRNA.


202968_s_at
0.0001551
2.3
DYRK2
dual-specificity tyrosine-(Y)-
12q15






phosphorylation regulated






kinase 2 (DYRK2), transcript






variant 1, mRNA.


202975_s_at
2.87E−05
−3.6
RHOBTB3
Rho-related BTB domain
5q15






containing 3 (RHOBTB3),






mRNA.


202992_at
0.000519
−5.4
C7
complement component 7 (C7),
5p13






mRNA.


202998_s_at
0.0006468
3
LOXL2
lysyl oxidase-like 2 (LOXL2),
8p21.3-p21.2






mRNA.


203088_at
0.0008436
−4.5
FBLN5
fibulin 5 (FBLN5), mRNA.
14q32.1


203156_at
3.37E−05
−2.4
AKAP11
A kinase (PRKA) anchor protein
13q14.11






11 (AKAP11), transcript variant






1, mRNA.


203166_at
0.0001939
−2.3
CFDP1
craniofacial development protein
16q22.2-q22.3






1 (CFDP1), mRNA.


203178_at
0.0002428
−3.3
GATM
glycine amidinotransferase (L-
15q21.1






arginine:glycine






amidinotransferase) (GATM),






mRNA.


203249_at
0.0004075
−2.5
EZH1
enhancer of zeste homolog 1
17q21.1-q21.3






(Drosophila) (EZH1), mRNA.


203297_s_at
4.33E−05
2.4
JARID2
Jumonji, AT rich interactive
6p24-p23






domain 2 (JARID2), mRNA.


203298_s_at
0.0005468
2.1
JARID2
Jumonji, AT rich interactive
6p24-p23






domain 2 (JARID2), mRNA.


203349_s_at
7.26E−05
2.8
ETV5
ets variant gene 5 (ets-related
3q28






molecule) (ETV5), mRNA.


203356_at
7.77E−05
−2.6
CAPN7
calpain 7 (CAPN7), mRNA.
3p24


203358_s_at
2.44E−05
2.9
EZH2
enhancer of zeste homolog 2
7q35-q36






(Drosophila) (EZH2), transcript






variant 2, mRNA.


203401_at
0.0003991
−3.8
PRPS2
phosphoribosyl pyrophosphate
Xp22.3-p22.2






synthetase 2 (PRPS2), mRNA.


203423_at
0.0008061
−3.9
RBP1
retinol binding protein 1,
3q23






cellular (RBP1), mRNA.


203424_s_at
0.0007037
−3.8
IGFBP5
insulin-like growth factor
2q33-q36






binding protein 5 (IGFBP5),






mRNA.


203427_at
7.05E−05
−2.6
ASF1A
ASF1 anti-silencing function 1
6q22.31






homolog A (S. cerevisiae)






(ASF1A), mRNA.


203450_at
0.0007322
−2.1
PGEA1
PKD2 interactor, golgi and
22q12






endoplasmic reticulum






associated 1 (PGEA1), transcript






variant 1, mRNA.


203455_s_at
0.0005583
2
SAT
spermidine/spermine N1-
Xp22.1






acetyltransferase (SAT), mRNA.


203459_s_at
8.97E−05
2.1
VPS16
vacuolar protein sorting 16
20p13-p12






(yeast) (VPS16), transcript






variant 2, mRNA.


203468_at
0.0009911
2.3
CDK10
cyclin-dependent kinase (CDC2-
16q24






like) 10 (CDK10), transcript






variant 2, mRNA.


203476_at
1.65E−05
2.6
TPBG
trophoblast glycoprotein
6q14-q15






(TPBG), mRNA.


203493_s_at
0.0009017
−2.1
PIG8
translokin (PIG8), mRNA.
11q21


203494_s_at
0.0001224
−2.2
PIG8
translokin (PIG8), mRNA.
11q21


203505_at
0.0001234
2.7
ABCA1
ATP-binding cassette, sub-
9q31.1






family A (ABC1), member 1


203549_s_at
0.0001406
3.9
LPL
lipoprotein lipase (LPL),
8p22






mRNA.


203599_s_at
0.0004137
−2
WBP4
WW domain binding protein 4
13q14.11






(formin binding protein 21)






(WBP4), mRNA.


203640_at
0.0002347
−2
MBNL2
muscleblind-like 2 (Drosophila)
13q32.1






(MBNL2), transcript variant 3,






mRNA.


203657_s_at
0.0007798
−2.3
CTSF
cathepsin F (CTSF), mRNA.
11q13


203680_at
9.00E−07
−6.1
PRKAR2B
protein kinase, cAMP-
7q22






dependent, regulatory, type II,






beta (PRKAR2B), mRNA.


203692_s_at
0.0005974
2.2
E2F3
E2F transcription factor 3
6p22






(E2F3), mRNA.


203695_s_at
0.0009082
−2.2
DFNA5
deafness, autosomal dominant 5
7p15






(DFNA5), mRNA.


203758_at
0.0002672
−2
CTSO
cathepsin O (CTSO), mRNA.
4q31-q32


203762_s_at
0.0003113
−2.1
D2LIC
dynein 2 light intermediate chain
2p25.1-p24.1






(D2LIC), transcript variant 1,






mRNA.


203799_at
1.06E−05
−3.3
CD302
CD302 antigen (CD302),
2q24.2






mRNA.


203803_at
3.79E−05
−3.9
PCYOX1
prenylcysteine oxidase 1
2p13.3






(PCYOX1), mRNA.


203845_at
0.0001562
−2.8
PCAF
p300/CBP-associated factor
3p24






(PCAF), mRNA.


203878_s_at
2.11E−05
3.7
MMP11
matrix metallopeptidase 11
22q11.23






(stromelysin 3) (MMP11),






mRNA.


203888_at
0.0008536
−4
THBD
thrombomodulin (THBD),
20p12-cen






mRNA.


203897_at
7.35E−05
−2.2
LOC57149
hypothetical protein A-211C6.1
16p11.2






(LOC57149), mRNA.


203908_at
0.0008304
2.7
SLC4A4
solute carrier family 4, sodium
4q21






bicarbonate cotransporter,






member 4 (SLC4A4), mRNA.


203936_s_at
0.0002019
9.4
MMP9
matrix metallopeptidase 9
20q11.2-q13.1






(gelatinase B, 92 kDa gelatinase,






92 kDa type IV collagenase)






(MMP9), mRNA.


204004_at
1.50E−06
−3.2
PAWR
PRKC, apoptosis, WT1,
12q21






regulator


204029_at
0.0005472
2.4
CELSR2
cadherin, EGF LAG seven-pass
1p21






G-type receptor 2 (flamingo






homolog, Drosophila)






(CELSR2), mRNA.


204041_at
0.0003564
−6.4
MAOB
Monoamine oxidase B
Xp11.23


204045_at
2.00E−07
−4.5
TCEAL1
Transcription elongation factor
Xq22.1






A (SII)-like 1


204078_at
0.0001645
2.6
SC65
synaptonemal complex protein
17q21.2






SC65 (SC65), mRNA.


204082_at
0.0002318
−2.8
PBX3
pre-B-cell leukemia
9q33-q34






transcription factor 3 (PBX3),






mRNA.


204109_s_at
0.0006487
2.5
NFYA
nuclear transcription factor Y,
6p21.3






alpha (NFYA), transcript variant






2, mRNA.


204136_at
7.18E−05
3.4
COL7A1
collagen, type VII, alpha 1
3p21.1






(epidermolysis bullosa,






dystrophic, dominant and






recessive) (COL7A1), mRNA.


204184_s_at
9.26E−05
3
ADRBK2
adrenergic, beta, receptor kinase
22q12.1






2 (ADRBK2), mRNA.


204235_s_at
2.45E−05
−4.3
GULP1
GULP, engulfment adaptor PTB
2q32.3-q33






domain containing 1 (GULP1),






mRNA.


204237_at
1.32E−05
−3.2
GULP1
GULP, engulfment adaptor PTB
2q32.3-q33






domain containing 1 (GULP1),






mRNA.


204256_at
0.0008289
2.1
ELOVL6
ELOVL family member 6,
4q25






elongation of long chain fatty






acids (FEN1/Elo2, SUR4/Elo3-






like, yeast) (ELOVL6), mRNA.


204285_s_at
1.20E−06
7.7
PMAIP1
phorbol-12-myristate-13-
18q21.32






acetate-induced protein 1






(PMAIP1), mRNA.


204286_s_at
3.50E−06
8.5
PMAIP1
phorbol-12-myristate-13-
18q21.32






acetate-induced protein 1






(PMAIP1), mRNA.


204387_x_at
7.88E−05
2.6
MRP63
mitochondrial ribosomal protein
13p11.1-q11






63 (MRP63), nuclear gene






encoding mitochondrial protein,






mRNA.


204454_at
0.0002053
−2.6
LDOC1
leucine zipper, down-regulated
Xq27






in cancer 1 (LDOC1), mRNA.


204473_s_at
0.0009569
2
ZNF592
zinc finger protein 592
15q25.3






(ZNF592), mRNA.


204493_at
0.0003505
2.7
BID
BH3 interacting domain death
22q11.1






agonist (BID), transcript variant






3, mRNA.


204495_s_at
0.0006319
2.8
DKFZP434H132
DKFZP434H132 protein
15q23


204531_s_at
0.0004972
2.5
BRCA1
breast cancer 1, early onset
17q21






(BRCA1), transcript variant






BRCA1-delta9-11, mRNA.


204595_s_at
1.40E−06
10.5
STC1
stanniocalcin 1 (STC1), mRNA.
8p21-p11.2


204597_x_at
0.0001207
7.5
STC1
stanniocalcin 1 (STC1), mRNA.
8p21-p11.2


204619_s_at
0.0005599
4.6
CSPG2
Chondroitin sulfate
5q14.3






proteoglycan 2 (versican)


204639_at
1.47E−05
4.2
ADA
adenosine deaminase (ADA),
20q12-q13.11






mRNA.


204641_at
0.0006499
4.6
NEK2
NIMA (never in mitosis gene a)-
1q32.2-q41






related kinase 2 (NEK2),






mRNA.


204669_s_at
0.0008759
2
RNF24
ring finger protein 24 (RNF24),
20p13-p12.1






mRNA.


204731_at
0.0002926
−3.2
TGFBR3
transforming growth factor, beta
1p33-p32






receptor III (betaglycan,






300 kDa) (TGFBR3), mRNA.


204735_at
3.38E−05
3.5
PDE4A
phosphodiesterase 4A, cAMP-
19p13.2






specific (phosphodiesterase E2






dunce homolog, Drosophila)






(PDE4A), mRNA.


204749_at
0.0005462
−3
NAP1L3
nucleosome assembly protein 1-
Xq21.3-q22






like 3 (NAP1L3), mRNA.


204786_s_at
1.59E−05
2.8
IFNAR2
interferon (alpha, beta and
21q22.11






omega) receptor 2 (IFNAR2),






transcript variant 1, mRNA.


204793_at
1.31E−05
−4.2
GPRASP1
G protein-coupled receptor
Xq22.1






associated sorting protein 1






(GPRASP1), mRNA.


204939_s_at
0.0007407
−5.7
PLN
phospholamban (PLN), mRNA.
6q22.1


204994_at
4.91E−05
4.5
MX2
myxovirus (influenza virus)
21q22.3






resistance 2 (mouse) (MX2),






mRNA.


205068_s_at
2.40E−06
3.8
ARHGAP26
Rho GTPase activating protein
5q31






26 (ARHGAP26), mRNA.


205079_s_at
0.0003985
−2
MPDZ
multiple PDZ domain protein
9p24-p22






(MPDZ), mRNA.


205226_at
0.0004657
−3
PDGFRL
platelet-derived growth factor
8p22-p21.3






receptor-like (PDGFRL),






mRNA.


205231_s_at
0.0008847
−2
EPM2A
epilepsy, progressive myoclonus
6q24






type 2A, Lafora disease (laforin)






(EPM2A), transcript variant 1,






mRNA.


205241_at
4.20E−05
4.5
SCO2
SCO cytochrome oxidase
22q13.33






deficient homolog 2 (yeast)






(SCO2), nuclear gene encoding






mitochondrial protein, mRNA.


205259_at
0.0002039
−2.5
NR3C2
nuclear receptor subfamily 3,
4q31.1






group C, member 2 (NR3C2),






mRNA.


205269_at
4.00E−06
8.7
LCP2
lymphocyte cytosolic protein 2
5q33.1-qter






(SH2 domain containing






leukocyte protein of 76 kDa)






(LCP2), mRNA.


205270_s_at
0.0006743
6.7
LCP2
lymphocyte cytosolic protein 2
5q33.1-qter






(SH2 domain containing






leukocyte protein of 76 kDa)






(LCP2), mRNA.


205304_s_at
0.0007499
2.4
KCNJ8
potassium inwardly-rectifying
12p11.23






channel, subfamily J, member 8






(KCNJ8), mRNA.


205353_s_at
3.92E−05
−3.3
PBP
prostatic binding protein (PBP),
12q24.23






mRNA.


205367_at
0.0001487
2.7
APS
adaptor protein with pleckstrin
7q22






homology and src homology 2






domains (APS), mRNA.


205370_x_at
0.0004381
2.9
DBT
dihydrolipoamide branched
1p31






chain transacylase E2 (DBT),






mRNA.


205381_at
0.0001503
−5.4
LRRC17
leucine rich repeat containing 17
7q22.1






(LRRC17), transcript variant 1,






mRNA.


205406_s_at
3.91E−05
2.5
SPA17
sperm autoantigenic protein 17
11q24.2






(SPA17), mRNA.


205412_at
6.30E−06
−2.7
ACAT1
acetyl-Coenzyme A
11q22.3-q23.1






acetyltransferase 1 (acetoacetyl






Coenzyme A thiolase)






(ACAT1), nuclear gene






encoding mitochondrial protein,






mRNA.


205463_s_at
0.000255
3.1
PDGFA
Platelet-derived growth factor
7p22






alpha polypeptide


205466_s_at
6.17E−05
−4.7
HS3ST1
heparan sulfate (glucosamine) 3-
4p16






O-sulfotransferase 1 (HS3ST1),






mRNA.


205479_s_at
0.000306
3.8
PLAU
plasminogen activator,
10q24






urokinase (PLAU), mRNA.


205483_s_at
0.0001248
6.9
G1P2
interferon, alpha-inducible
1p36.33






protein (clone IFI-15K) (G1P2),






mRNA.


205532_s_at
0.0004946
2.8
CDH6
cadherin 6, type 2, K-cadherin
5p15.1-p14






(fetal kidney) (CDH6), mRNA.


205572_at
0.0003915
5.4
ANGPT2
angiopoietin 2 (ANGPT2),
8p23.1






mRNA.


205687_at
0.000463
2
UBPH
similar to ubiquitin binding
16p12






protein (UBPH), mRNA.


205771_s_at
0.0009058
−2.3
AKAP7
A kinase (PRKA) anchor protein
6q23






7 (AKAP7), transcript variant






alpha, mRNA.


205812_s_at
9.00E−06
2.4
TMED9
transmembrane emp24 protein
5q35.3






transport domain containing 9






(TMED9), mRNA.


205849_s_at
0.0005492
−2
UQCRB
ubiquinol-cytochrome c
8q22






reductase binding protein






(UQCRB), mRNA.


205862_at
7.65E−05
−8.6
GREB1
GREB1 protein (GREB1),
2p25.1






transcript variant a, mRNA.


205871_at
0.0003369
3.1
PLGLB1
plasminogen-like B1 (PLGLB1),
2






mRNA.


205943_at
0.0004032
4.4
TDO2
tryptophan 2,3-dioxygenase
4q31-q32






(TDO2), mRNA.


205961_s_at
0.0002574
−2.9
PSIP1
PC4 and SFRS1 interacting
9p22.3






protein 1 (PSIP1), transcript






variant 2, mRNA.


206026_s_at
p < 1e−07
29.1
TNFAIP6
tumor necrosis factor, alpha-
2q23.3






induced protein 6 (TNFAIP6),






mRNA.


206158_s_at
0.0006831
−2.2
ZNF9
zinc finger protein 9 (a cellular
3q21






retroviral nucleic acid binding






protein) (ZNF9), mRNA.


206169_x_at
0.0008428
3.9
RoXaN
Zinc finger CCCH-type
22q13.2






containing 7B


206211_at
0.0002057
−8.9
SELE
selectin E (endothelial adhesion
1q22-q25






molecule 1) (SELE), mRNA.


206247_at
9.38E−05
3.3
MICB
MHC class I polypeptide-related
6p21.3






sequence B (MICB), mRNA.


206359_at
0.0004232
−3.5
SOCS3
Suppressor of cytokine signaling 3
17q25.3


206377_at
2.40E−06
6
FOXF2
forkhead box F2 (FOXF2),
6p25.3






mRNA.


206435_at
0.0009321
3.2
GALGT
UDP-N-acetyl-alpha-D-
12q13.3






galactosamine:(N-






acetylneuraminyl)-






galactosylglucosylceramide N-






acetylgalactosaminyltransferase






(GalNAc-T) (GALGT), mRNA.


206483_at
0.0009412
2
LRRC6
leucine rich repeat containing 6
8q24.22






(LRRC6), mRNA.


206551_x_at
0.0001129
2.7
DRE1
DRE1 protein
3q27.1


206571_s_at
0.000131
2.7
MAP4K4
mitogen-activated protein kinase
2q11.2-q12






kinase kinase kinase 4






(MAP4K4), transcript variant 1,






mRNA.


206621_s_at
0.0002653
−2.3
WBSCR1
Williams-Beuren syndrome
7q11.23






chromosome region 1






(WBSCR1), transcript variant 2,






mRNA.


206637_at
3.52E−05
4.8
P2RY14
purinergic receptor P2Y, G-
3q21-q25






protein coupled, 14 (P2RY14),






mRNA.


206792_x_at
6.82E−05
3.7
PDE4C
phosphodiesterase 4C, cAMP-
19p13.11






specific (phosphodiesterase E1






dunce homolog, Drosophila)






(PDE4C), mRNA.


206809_s_at
0.0004239
−2.8
HNRPA3P1
Heterogeneous nuclear
10q11.21






ribonucleoprotein A3






pseudogene 1


206857_s_at
0.0002413
2.5
FKBP1B
FK506 binding protein 1B, 12.6 kDa
2p23.3






(FKBP1B), transcript






variant 1, mRNA.


206874_s_at
9.71E−05
−2.9
SLK
STE20-like kinase (yeast)
10q25.1


206927_s_at
0.0004319
3.3
GUCY1A2
guanylate cyclase 1, soluble,
11q21-q22






alpha 2 (GUCY1A2), mRNA.


207040_s_at
1.56E−05
−2.6
ST13
suppression of tumorigenicity 13
22q13.2






(colon carcinoma) (Hsp70






interacting protein) (ST13),






mRNA.


207132_x_at
0.0002292
−2.5
PFDN5
prefoldin 5 (PFDN5), transcript
12q12






variant 1, mRNA.


207147_at
0.0007438
3.3
DLX2
distal-less homeo box 2 (DLX2),
2q32






mRNA.


207170_s_at
1.76E−05
−2.6
LETMD1
LETM1 domain containing 1
12q13.12






(LETMD1), transcript variant 3,






mRNA.


207239_s_at
0.0002121
2.2
PCTK1
PCTAIRE protein kinase 1
Xp11.3-p11.23






(PCTK1), transcript variant 1,






mRNA.


207365_x_at
6.06E−05
3.7
USP34
Ubiquitin specific protease 34
2p15


207386_at
0.0009685
2.7
CYP7B1
cytochrome P450, family 7,
8q21.3






subfamily B, polypeptide 1






(CYP7B1), mRNA.


207598_x_at
0.0001463
3.5
XRCC2
X-ray repair complementing
7q36.1






defective repair in Chinese






hamster cells 2 (XRCC2),






mRNA.


207688_s_at
0.0006478
2.4
LOC387933
PREDICTED: similar to
13






heterogeneous nuclear






ribonucleoprotein A3






(LOC387933), mRNA.


207730_x_at
0.0002746
3.1
FLJ20700
hypothetical protein FLJ20700
19p13.3


207761_s_at
1.38E−05
−4.2
DKFZP586A0522
DKFZP586A0522 protein
12q13.12






(DKFZP586A0522), mRNA.


207961_x_at
0.0006679
−3.8
MYH11
myosin, heavy polypeptide 11,
16p13.13-p13.12






smooth muscle (MYH11),






transcript variant SM1, mRNA.


207974_s_at
0.0001511
−2.1
SKP1A
S-phase kinase-associated
5q31






protein 1A (p19A) (SKP1A),






transcript variant 2, mRNA.


207983_s_at
0.0009431
−2.3
STAG2
stromal antigen 2 (STAG2),
Xq25






mRNA.


208092_s_at
0.0004178
2.9
FAM49A
family with sequence similarity
2p24.3-p24.2






49, member A (FAM49A),






mRNA.


208137_x_at
0.0008157
2.6
ZNF611
Zinc finger protein 611
19q13.41


208238_x_at
0.0007093
2.4
LZLP
leucine zipper-like protein
11q13.1


208246_x_at
8.09E−05
3.6
FLJ20006
hypothetical protein FLJ20006
16q23.1


208248_x_at
5.35E−05
−3.5
APLP2
amyloid beta (A4) precursor-like
11q24






protein 2 (APLP2), mRNA.


208540_x_at
0.0007507
2
S100A14
S100 calcium binding protein
7q22-q31.1






A14 (calgizzarin)


208626_s_at
0.000509
−2.1
VAT1
vesicle amine transport protein 1
17q21






homolog (T californica)






(VAT1), mRNA.


208631_s_at
0.0003804
−2.3
HADHA
hydroxyacyl-Coenzyme A
2p23






dehydrogenase/3-ketoacyl-






Coenzyme A thiolase/enoyl-






Coenzyme A hydratase






(trifunctional protein), alpha






subunit (HADHA), mRNA.


208635_x_at
8.57E−05
−2
NACA
nascent-polypeptide-associated
12q23-q24.1






complex alpha polypeptide






(NACA), mRNA.


208643_s_at
2.41E−05
−2.9
XRCC5
X-ray repair complementing
2q35






defective repair in Chinese






hamster cells 5 (double-strand-






break rejoining; Ku autoantigen,






80 kDa) (XRCC5), mRNA.


208647_at
0.0004172
−2.1
FDFT1
farnesyl-diphosphate
8p23.1-p22






farnesyltransferase 1 (FDFT1),






mRNA.


208653_s_at
0.0002993
3
CD164
CD164 antigen, sialomucin
6q21






(CD164), mRNA.


208655_at
0.0001893
−2.8
CCNI
Cyclin I
4q21.1


208658_at
0.0005959
2
PDIA4
protein disulfide isomerase
7q35






family A, member 4 (PDIA4),






mRNA.


208662_s_at
0.0009682
−2.1
TTC3
tetratricopeptide repeat domain
21q22.2






3 (TTC3), transcript variant 2,






mRNA.


208666_s_at
5.00E−07
−4.5
ST13
suppression of tumorigenicity 13
22q13.2






(colon carcinoma) (Hsp70






interacting protein) (ST13),






mRNA.


208667_s_at
4.53E−05
−2.8
ST13
suppression of tumorigenicity 13
22q13.2






(colon carcinoma) (Hsp70






interacting protein) (ST13),






mRNA.


208671_at
0.0005205
−2
TDE2
tumor differentially expressed 2
6q22.31






(TDE2), mRNA.


208673_s_at
0.0005001
−2.2
SFRS3
splicing factor, arginine/serine-
6p21






rich 3 (SFRS3), mRNA.


208697_s_at
4.85E−05
−2.4
EIF3S6
eukaryotic translation initiation
8q22-q23






factor 3, subunit 6 48 kDa






(EIF3S6), mRNA.


208703_s_at
0.0002231
−3.7
APLP2
amyloid beta (A4) precursor-like
11q24






protein 2 (APLP2), mRNA.


208704_x_at
4.90E−05
−3.6
APLP2
amyloid beta (A4) precursor-like
11q24






protein 2 (APLP2), mRNA.


208740_at
7.40E−05
−2.2
SAP18
sin3-associated polypeptide,
13q12.11






18 kDa (SAP18), mRNA.


208760_at
7.17E−05
−2.7
UBE2I
Ubiquitin-conjugating enzyme
16p13.3






E2I (UBC9 homolog, yeast)


208770_s_at
0.0005681
−2
EIF4EBP2
eukaryotic translation initiation
10q21-q22






factor 4E binding protein 2






(EIF4EBP2), mRNA.


208771_s_at
2.10E−05
−2.6
LTA4H
leukotriene A4 hydrolase
12q22






(LTA4H), mRNA.


208781_x_at
0.0006007
−2.2
SNX3
sorting nexin 3 (SNX3),
6q21






transcript variant 1, mRNA.


208791_at
0.0001028
−4.4
CLU
clusterin (complement lysis
8p21-p12






inhibitor, SP-40,40, sulfated






glycoprotein 2, testosterone-






repressed prostate message 2,






apolipoprotein J) (CLU),






transcript variant 1, mRNA.


208792_s_at
0.0001175
−4.5
CLU
clusterin (complement lysis
8p21-p12






inhibitor, SP-40,40, sulfated






glycoprotein 2, testosterone-






repressed prostate message 2,






apolipoprotein J) (CLU),






transcript variant 1, mRNA.


208794_s_at
1.47E−05
2.4
SMARCA4
SWI/SNF related, matrix
19p13.2






associated, actin dependent






regulator of chromatin,






subfamily a, member 4






(SMARCA4), mRNA.


208796_s_at
0.0001639
−2.6
CCNG1
cyclin G1 (CCNG1), transcript
5q32-q34






variant 2, mRNA.


208848_at
1.50E−06
−3.7
ADH5
alcohol dehydrogenase 5 (class
4q21-q25






III), chi polypeptide (ADH5),






mRNA.


208860_s_at
0.0001719
−2
ATRX
alpha thalassemia/mental
Xq13.1-q21.1






retardation syndrome X-linked






(RAD54 homolog, S. cerevisiae)






(ATRX), transcript variant 2,






mRNA.


208873_s_at
5.10E−05
−2.1
C5orf18
chromosome 5 open reading
5q22-q23






frame 18 (C5orf18), mRNA.


208920_at
0.0003111
−2.3
SRI
sorcin (SRI), transcript variant 2,
7q21.1






mRNA.


208925_at
0.0005429
−2.3
C3orf4
chromosome 3 open reading
3p11-q11






frame 4 (C3orf4), mRNA.


208950_s_at
0.0004293
−2.3
ALDH7A1
aldehyde dehydrogenase 7
5q31






family, member A1






(ALDH7A1), mRNA.


208951_at
4.20E−05
−2.8
ALDH7A1
aldehyde dehydrogenase 7
5q31






family, member A1






(ALDH7A1), mRNA.


208962_s_at
0.0003548
−2.4
FADS1
fatty acid desaturase 1 (FADS1),
11q12.2-q13.1






mRNA.


208990_s_at
5.40E−05
−2.2
HNRPH3
heterogeneous nuclear
10q22






ribonucleoprotein H3 (2H9)






(HNRPH3), transcript variant






2H9A, mRNA.


209009_at
1.50E−06
−3
ESD
esterase D/formylglutathione
13q14.1-q14.2






hydrolase (ESD), mRNA.


209030_s_at
3.07E−05
2.6
IGSF4
immunoglobulin superfamily,
11q23.2






member 4 (IGSF4), mRNA.


209034_at
3.20E−06
−4.5
PNRC1
proline-rich nuclear receptor
6q15






coactivator 1 (PNRC1), mRNA.


209068_at
1.00E−06
−3.5
HNRPDL
heterogeneous nuclear
4q13-q21






ribonucleoprotein D-like






(HNRPDL), transcript variant 2,






mRNA.


209081_s_at
8.30E−06
5
COL18A1
collagen, type XVIII, alpha 1
21q22.3






(COL18A1), transcript variant 2,






mRNA.


209082_s_at
4.04E−05
5.1
COL18A1
collagen, type XVIII, alpha 1
21q22.3






(COL18A1), transcript variant 2,






mRNA.


209137_s_at
0.0007917
−2.1
USP10
ubiquitin specific peptidase 10
16q24.1






(USP10), mRNA.


209143_s_at
0.0009179
−2.1
CLNS1A
chloride channel, nucleotide-
11q13.5-q14






sensitive, 1A (CLNS1A),






mRNA.


209146_at
0.0002317
−3.2
SC4MOL
sterol-C4-methyl oxidase-like
4q32-q34






(SC4MOL), transcript variant 2,






mRNA.


209169_at
0.0003677
4.2
GPM6B
glycoprotein M6B (GPM6B),
Xp22.2






transcript variant 1, mRNA.


209170_s_at
0.00014
5.8
GPM6B
glycoprotein M6B (GPM6B),
Xp22.2






transcript variant 4, mRNA.


209243_s_at
3.90E−06
−7.7
PEG3
paternally expressed 3 (PEG3),
19q13.4






mRNA.


209305_s_at
0.0001816
−3.7
GADD45B
growth arrest and DNA-damage-
19p13.3






inducible, beta (GADD45B),






mRNA.


209337_at
4.01E−05
−3
PSIP1
PC4 and SFRS1 interacting
9p22.3






protein 1 (PSIP1), transcript






variant 2, mRNA.


209357_at
5.76E−05
−3.3
CITED2
Cbp/p300-interacting
6q23.3






transactivator, with Glu/Asp-






rich carboxy-terminal domain, 2






(CITED2), mRNA.


209360_s_at
7.44E−05
4.8
RUNX1
runt-related transcription factor
21q22.3






1 (acute myeloid leukemia 1;






aml1 oncogene) (RUNX1),






transcript variant 1, mRNA.


209384_at
0.0002565
−2
PROSC
proline synthetase co-transcribed
8p11.2






homolog (bacterial) (PROSC),






mRNA.


209385_s_at
3.63E−05
−2.6
PROSC
proline synthetase co-transcribed
8p11.2






homolog (bacterial) (PROSC),






mRNA.


209447_at
3.84E−05
−2.5
SYNE1
spectrin repeat containing,
6q25






nuclear envelope 1 (SYNE1),






transcript variant alpha, mRNA.


209512_at
3.90E−06
−3.9
HSDL2
hydroxysteroid dehydrogenase
9q32






like 2 (HSDL2), mRNA.


209513_s_at
1.94E−05
−3.9
HSDL2
hydroxysteroid dehydrogenase
9q32






like 2 (HSDL2), mRNA.


209596_at
4.80E−06
6.9
MXRA5
matrix-remodelling associated 5
Xp22.33






(MXRA5), mRNA.


209605_at
0.0004157
−2.4
TST
thiosulfate sulfurtransferase
22q13.1






(rhodanese) (TST), nuclear gene






encoding mitochondrial protein,






mRNA.


209612_s_at
0.0001328
−4.5
ADH1B
alcohol dehydrogenase IB (class
4q21-q23






I), beta polypeptide (ADH1B),






mRNA.


209613_s_at
0.0003092
−5.8
ADH1B
alcohol dehydrogenase IB (class
4q21-q23






I), beta polypeptide (ADH1B),






mRNA.


209633_at
0.0006246
−2.2
PPP2R3A
protein phosphatase 2 (formerly
3q22.1






2A), regulatory subunit B′′,






alpha (PPP2R3A), transcript






variant 2, mRNA.


209657_s_at
0.0003701
−2.1
HSF2
heat shock transcription factor 2
6q22.31






(HSF2), mRNA.


209685_s_at
1.10E−05
3.7
PRKCB1
protein kinase C, beta 1
16p11.2






(PRKCB1), transcript variant 2,






mRNA.


209733_at
1.64E−05
−2.7
LOC286440
Hypothetical protein
Xq22.3






LOC286440


209737_at
0.0003955
−2.2
MAGI2
membrane associated guanylate
7q21






kinase, WW and PDZ domain






containing 2 (MAGI2), mRNA.


209875_s_at
9.40E−06
9.5
SPP1
secreted phosphoprotein 1
4q21-q25






(osteopontin, bone sialoprotein






I, early T-lymphocyte activation






1) (SPP1), mRNA.


209894_at
0.0001625
−4.2
LEPR
leptin receptor (LEPR),
1p31






transcript variant 2, mRNA.


209897_s_at
0.0001202
2.5
SLIT2
slit homolog 2 (Drosophila)
4p15.2






(SLIT2), mRNA.


209969_s_at
1.51E−05
3.7
STAT1
signal transducer and activator
2q32.2






of transcription 1, 91 kDa






(STAT1), transcript variant beta,






mRNA.


210048_at
0.0008484
2.2
NAPG
N-ethylmaleimide-sensitive
18p11.22






factor attachment protein,






gamma (NAPG), mRNA.


210069_at
3.07E−05
2.8
CPT1B
carnitine palmitoyltransferase
22q13.33






1B (muscle) (CPT1B), nuclear






gene encoding mitochondrial






protein, transcript variant 3,






mRNA.


210365_at
0.0002181
2.6
RUNX1
Runt-related transcription factor
21q22.3






1 (acute myeloid leukemia 1;






aml1 oncogene)


210438_x_at
0.0003684
−2.7
TROVE2
TROVE domain family,
1q31






member 2 (TROVE2), mRNA.


210621_s_at
0.0004078
−2.1
RASA1
RAS p21 protein activator
5q13.3






(GTPase activating protein) 1






(RASA1), transcript variant 2,






mRNA.


210664_s_at
0.0006973
2.4
TFPI
tissue factor pathway inhibitor
2q31-q32.1






(lipoprotein-associated






coagulation inhibitor) (TFPI),






transcript variant 1, mRNA.


210665_at
0.0003981
3.1
TFPI
tissue factor pathway inhibitor
2q31-q32.1






(lipoprotein-associated






coagulation inhibitor) (TFPI),






transcript variant 2, mRNA.


210679_x_at
0.000265
3.4
BCL7A
B-cell CLL/lymphoma 7A
12q24.13


210788_s_at
1.26E−05
−3.1
DHRS7
dehydrogenase/reductase (SDR
14q23.1






family) member 7 (DHRS7),






mRNA.


210800_at
0.0009706
4.7
MGC12262
hypothetical protein MGC12262


210809_s_at
0.0002365
6.7
POSTN
periostin, osteoblast specific
13q13.3






factor (POSTN), mRNA.


210944_s_at
0.0009253
2.3
CAPN3
calpain 3, (p94) (CAPN3),
15q15.1-q21.1






transcript variant 7, mRNA.


210950_s_at
1.38E−05
−3.3
FDFT1
farnesyl-diphosphate
8p23.1-p22






farnesyltransferase 1 (FDFT1),






mRNA.


211040_x_at
0.0006364
2.4
GTSE1
G-2 and S-phase expressed 1
22q13.2-q13.3






(GTSE1), mRNA.


211276_at
8.52E−05
−5.6
TCEAL2
transcription elongation factor A
Xq22.1-q22.3






(SII)-like 2 (TCEAL2), mRNA.


211423_s_at
0.000164
−2.8
SCSDL
sterol-C5-desaturase (ERG3
11q23.3






delta-5-desaturase homolog,






fungal)-like (SC5DL), transcript






variant 1, mRNA.


211445_x_at
0.0007153
3.6
FKSG17
FKSG17
8q22.3


211452_x_at
5.23E−05
3.4
LRRFIP1
leucine rich repeat (in FLII)
2q37.3






interacting protein 1 (LRRFIP1),






mRNA.


211454_x_at
0.0006917
3.7


211569_s_at
2.66E−05
−5.7
HADHSC
L-3-hydroxyacyl-Coenzyme A
4q22-q26






dehydrogenase, short chain






(HADHSC), mRNA.


211597_s_at
1.00E−07
13.1
HOP
homeodomain-only protein
4q11-q12






(HOP), transcript variant 2,






mRNA.


211623_s_at
0.0008386
−2.2
FBL
fibrillarin (FBL), mRNA.
19q13.1


211666_x_at
9.60E−06
−2.6
RPL3
ribosomal protein L3 (RPL3),
22q13






mRNA.


211673_s_at
5.65E−05
3.5
MOCS1
Molybdenum cofactor synthesis 1
6p21.3


211710_x_at
0.0001136
−2.2
RPL4
ribosomal protein L4 (RPL4),
15q22






mRNA.


211725_s_at
0.0006346
2.4
BID
BH3 interacting domain death
22q11.1






agonist (BID), transcript variant






3, mRNA.


211727_s_at
0.0004584
−2.5
COX11
COX11 homolog, cytochrome c
17q22






oxidase assembly protein (yeast)






(COX11), nuclear gene






encoding mitochondrial protein,






mRNA.


211749_s_at
7.41E−05
−2.6
VAMP3
vesicle-associated membrane
1p36.23






protein 3 (cellubrevin)






(VAMP3), mRNA.


211762_s_at
0.0002377
2.3
KPNA2
karyopherin alpha 2 (RAG
17q23.1-q23.3






cohort 1, importin alpha 1)






(KPNA2), mRNA.


211769_x_at
4.67E−05
−2.4
TDE1
tumor differentially expressed 1
20q13.1-13.3






(TDE1), transcript variant 1,






mRNA.


211813_x_at
1.69E−05
−4.8
DCN
decorin (DCN), transcript
12q21.33






variant D, mRNA.


211896_s_at
0.0006287
−3.7
DCN
decorin (DCN), transcript
12q21.33






variant C, mRNA.


211937_at
0.0001012
−3.2
EIF4B
eukaryotic translation initiation
12q13.13






factor 4B (EIF4B), mRNA.


211938_at
3.00E−07
−3.2
EIF4B
eukaryotic translation initiation
12q13.13






factor 4B (EIF4B), mRNA.


211941_s_at
2.01E−05
−2.3
PBP
prostatic binding protein (PBP),
12q24.23






mRNA.


211942_x_at
7.02E−05
−2.8
RPL13A
Ribosomal protein L13a
19q13.3


211964_at
0.0006935
3.4
COL4A2
Collagen, type IV, alpha 2
13q34


211980_at
0.0005493
3.9
COL4A1
collagen, type IV, alpha 1
13q34






(COL4A1), mRNA.


211986_at
3.42E−05
−3.3
AHNAK
AHNAK nucleoprotein
11q12.2






(desmoyokin) (AHNAK),






transcript variant 1, mRNA.


211988_at
1.30E−05
−2.2
SMARCE1
SWI/SNF related, matrix
17q21.2






associated, actin dependent






regulator of chromatin,






subfamily e, member 1






(SMARCE1), mRNA.


211994_at
0.0001256
−2

Transcribed locus, strongly
12






similar to XP_508919.1






PREDICTED: similar to protein






kinase, lysine deficient 1; kinase






deficient protein [Pan






troglodytes]


211997_x_at
5.21E−05
−2.5
H3F3B
H3 histone, family 3B (H3.3B)
17q25






(H3F3B), mRNA.


211998_at
7.80E−05
−3
H3F3B
H3 histone, family 3B (H3.3B)
17q25






(H3F3B), mRNA.


212037_at
0.0001271
−2.7
PNN
Pinin, desmosome associated
14q21.1






protein


212044_s_at
9.00E−07
3.4
RPL27A
ribosomal protein L27a
11p15






(RPL27A), mRNA.


212052_s_at
0.0005363
2
KIAA0676
KIAA0676 protein
5q35.3






(KIAA0676), transcript variant






2, mRNA.


212094_at
0.0001189
−4.8
PEG10
PREDICTED: paternally
7






expressed 10 (PEG10), mRNA.


212096_s_at
0.0002831
−2.7
MTUS1
mitochondrial tumor suppressor
8p22






1 (MTUS1), nuclear gene






encoding mitochondrial protein,






transcript variant 5, mRNA.


212131_at
4.28E−05
−2
FAM61A
family with sequence similarity
19q13.11






61, member A (FAM61A),






mRNA.


212134_at
0.0003033
2
PHLDB1
pleckstrin homology-like
11q23.3






domain, family B, member 1






(PHLDB1), mRNA.


212144_at
0.0005431
−2.1
UNC84B
unc-84 homolog B (C. elegans)
22q13.1






(UNC84B), mRNA.


212148_at
9.20E−06
−2.9
PBX1
Pre-B-cell leukemia
1q23






transcription factor 1


212151_at
0.0001748
−2.5
PBX1
Pre-B-cell leukemia
1q23






transcription factor 1


212171_x_at
0.0006474
3.3
VEGF
vascular endothelial growth
6p12






factor (VEGF), transcript variant






6, mRNA.


212179_at
0.0003184
−2.5
C6orf111
Chromosome 6 open reading
6q16.3






frame 111


212188_at
0.000184
−2.9
KCTD12
potassium channel
13q22.3






tetramerisation domain






containing 12 (KCTD12),






mRNA.


212195_at
0.0002328
−2.1
IL6ST
Interleukin 6 signal transducer
5q11






(gp130, oncostatin M receptor)


212199_at
0.0001637
−2.5
MRFAP1L1
Morf4 family associated protein
4p16.1






1-like 1 (MRFAP1L1),






transcript variant 2, mRNA.


212215_at
0.0001021
−2.8
PREPL
prolyl endopeptidase-like
2p22.1






(PREPL), mRNA.


212224_at
1.18E−05
−4.3
ALDH1A1
aldehyde dehydrogenase 1
9q21.13






family, member A1






(ALDH1A1), mRNA.


212236_x_at
0.0002768
5.3
KRT17
keratin 17 (KRT17), mRNA.
17q12-q21


212254_s_at
4.00E−06
−2.7
DST
dystonin (DST), transcript
6p12-p11






variant 1eA, mRNA.


212256_at
0.0007645
−2.9
GALNT10
UDP-N-acetyl-alpha-D-
5q33.2






galactosamine:polypeptide N-






acetylgalactosaminyltransferase






10 (GalNAc-T10)


212323_s_at
2.11E−05
2.5
VPS13D
vacuolar protein sorting 13D
1p36.22-p36.21






(yeast) (VPS13D), transcript






variant 2, mRNA.


212351_at
8.00E−06
2.2
EIF2B5
eukaryotic translation initiation
3q27.1






factor 2B, subunit 5 epsilon,






82 kDa (EIF2B5), mRNA.


212353_at
0.000225
3.6
SULF1
sulfatase 1 (SULF1), mRNA.
8q13.2-q13.3


212354_at
0.0002219
2.9
SULF1
sulfatase 1 (SULF1), mRNA.
8q13.2-q13.3


212365_at
0.0003347
4.1
MYO1B
myosin IB (MYO1B), mRNA.
2q12-q34


212368_at
0.0002867
−2.3
ZNF292
PREDICTED: zinc finger
6






protein 292 (ZNF292), mRNA.


212408_at
1.41E−05
−2.6
TOR1AIP1
torsin A interacting protein 1
1q24.2






(TOR1AIP1), mRNA.


212413_at
0.0005848
2
6-Sep
septin 6 (SEPT6), transcript
Xq24






variant II, mRNA.


212414_s_at
0.0001527
2.4
6-Sep
septin 6 (SEPT6), transcript
Xq24






variant II, mRNA.


212435_at
0.0003489
−2.3
TRIM33
tripartite motif-containing 33
1p13.1






(TRIM33), transcript variant b,






mRNA.


212468_at
0.0001247
−2
SPAG9
Sperm associated antigen 9
17q21.33


212498_at
1.50E−06
−2.7
MARCH-VI
Membrane-associated ring
5p15.2






finger (C3HC4) 6


212510_at
0.0004844
−3.1
GPD1L
glycerol-3-phosphate
3p24.1






dehydrogenase 1-like (GPD1L),






mRNA.


212520_s_at
3.25E−05
3.1
SMARCA4
SWI/SNF related, matrix
19p13.2






associated, actin dependent






regulator of chromatin,






subfamily a, member 4






(SMARCA4), mRNA.


212526_at
6.11E−05
−2.3
SPG20
spastic paraplegia 20, spartin
13q13.3






(Troyer syndrome) (SPG20),






mRNA.


212549_at
6.64E−05
−2.4
STAT5B
signal transducer and activator
17q11.2






of transcription 5B (STAT5B),






mRNA.


212556_at
0.0003565
2.8
SCRIB
scribbled homolog (Drosophila)
8q24.3






(SCRIB), transcript variant 2,






mRNA.


212586_at
0.0004909
−2.1
CAST
calpastatin (CAST), transcript
5q15-q21






variant 2, mRNA.


212595_s_at
0.0007053
−2
DAZAP2
DAZ associated protein 2
12q12






(DAZAP2), mRNA.


212609_s_at
1.85E−05
−2.4
AKT3
V-akt murine thymoma viral
1q43-q44






oncogene homolog 3 (protein






kinase B, gamma)


212624_s_at
5.40E−06
6.9
CHN1
chimerin (chimaerin) 1 (CHN1),
2q31-q32.1






transcript variant 2, mRNA.


212632_at
0.0008287
−2
STX7
Syntaxin 7
6q23.1


212638_s_at
0.0007524
−2.2
WWP1
WW domain containing E3
8q21






ubiquitin protein ligase 1






(WWP1), mRNA.


212644_s_at
0.0001184
−2.3
C14orf32
chromosome 14 open reading
14q22.2-q22.3






frame 32 (C14orf32), mRNA.


212646_at
0.0003295
2.6
RAFTLIN
raft-linking protein (RAFTLIN),
3p25.1-p24.3






mRNA.


212653_s_at
3.00E−07
−3.8
EHBP1
EH domain binding protein 1
2p15






(EHBP1), mRNA.


212675_s_at
0.0003318
−3.5
KIAA0582
KIAA0582
2p14


212730_at
0.0006706
−3.4
DMN
desmuslin (DMN), transcript
15p26.3






variant B, mRNA.


212731_at
0.000105
−3.3
ANKRD46
ankyrin repeat domain 46
8q22.3






(ANKRD46), mRNA.


212751_at
0.0003336
−2.7
UBE2N
ubiquitin-conjugating enzyme
12q22






E2N (UBC13 homolog, yeast)






(UBE2N), mRNA.


212769_at
0.000184
2.7
TLE3
Transducin-like enhancer of
15q22






split 3 (E(sp1) homolog,






Drosophila)


212776_s_at
0.0004769
−3
KIAA0657
PREDICTED: KIAA0657
2






protein (KIAA0657), mRNA.


212779_at
0.0005762
−2
KIAA1109
PREDICTED: hypothetical
4






protein KIAA1109






(KIAA1109), mRNA.


212798_s_at
3.32E−05
−2
ANKMY2
ankyrin repeat and MYND
7p21






domain containing 2






(ANKMY2), mRNA.


212809_at
7.32E−05
2.6
NFATC2IP
nuclear factor of activated T-
16p11.2






cells, cytoplasmic, calcineurin-






dependent 2 interacting protein






(NFATC2IP), mRNA.


212841_s_at
0.0004662
2.6
PPFIBP2
PTPRF interacting protein,
11p15.4






binding protein 2 (liprin beta 2)






(PPFIBP2), mRNA.


212943_at
0.0009511
−2.2
KIAA0528
KIAA0528 gene product
12p12.1






(KIAA0528), mRNA.


212971_at
0.0004892
2.2
CARS
Cysteinyl-tRNA synthetase
11p15.5


213002_at
7.97E−05
3.8
MARCKS
myristoylated alanine-rich
6q22.2






protein kinase C substrate






(MARCKS), mRNA.


213005_s_at
0.0003887
−2.3
ANKRD15
ankyrin repeat domain 15
9p24.3






(ANKRD15), transcript variant






1, mRNA.


213027_at
0.0001503
−2.1
SSA2
TROVE domain family,
1q31






member 2


213029_at
6.18E−05
−2.4
NFIB
Nuclear factor I/B
9p24.1


213047_x_at
0.0003826
−2.6
SET
SET translocation (myeloid
9q34






leukemia-associated) (SET),






mRNA.


213074_at
0.0002338
−2.6
PHIP
Pleckstrin homology domain
6q14






interacting protein


213085_s_at
0.0005289
3.8
KIBRA
KIBRA protein (KIBRA),
5q34






mRNA.


213093_at
1.80E−05
−2.8
PRKCA
protein kinase C, alpha
17q22-q23.2






(PRKCA), mRNA.


213110_s_at
5.00E−06
−3.9
COL4A5
collagen, type IV, alpha 5
Xq22






(Alport syndrome) (COL4A5),






transcript variant 1, mRNA.


213111_at
0.0006152
−2
PIP5K3
phosphatidylinositol-3-
2q34






phosphate/phosphatidylinositol






5-kinase, type III (PIP5K3),






transcript variant 1, mRNA.


213117_at
0.0008783
−2
KLHL9
kelch-like 9 (Drosophila)
9p22






(KLHL9), mRNA.


213139_at
0.0008306
2.6
SNAI2
snail homolog 2 (Drosophila)
8q11






(SNAI2), mRNA.


213146_at
0.000125
2.9
KIAA0346
KIAA0346 protein
17p13.1


213224_s_at
0.0005954
−2
LOC92482
PREDICTED: hypothetical
10






protein LOC92482






(LOC92482), mRNA.


213227_at
0.0001677
−2
PGRMC2
Progesterone receptor membrane
4q26






component 2


213248_at
0.0006637
2.4
LOC221362
Hypothetical protein
6p12.3






LOC221362


213258_at
3.10E−05
2.5
TFPI
Tissue factor pathway inhibitor
2q31-q32.1






(lipoprotein-associated






coagulation inhibitor)


213272_s_at
6.80E−06
−4
LOC57146
promethin (LOC57146), mRNA.
16p12


213344_s_at
3.91E−05
2.7
H2AFX
H2A histone family, member X
11q23.2-q23.3






(H2AFX), mRNA.


213350_at
1.20E−05
6.1
RPS11
ribosomal protein S11 (RPS11),
19q13.3






mRNA.


213364_s_at
4.00E−07
−3.7
SNX1
sorting nexin 1 (SNX1),
15q22.31






transcript variant 2, mRNA.


213370_s_at
0.0001347
2.1
SFMBT1
Scm-like with four mbt domains
3p21.1






1 (SFMBT1), transcript variant






3, mRNA.


213397_x_at
4.30E−06
−5.2
RNASE4
ribonuclease, RNase A family, 4
14q11.1






(RNASE4), transcript variant 3,






mRNA.


213405_at
0.000663
−2.4
RAB22A
RAB22A, member RAS
20q13.32






oncogene family (RAB22A),






mRNA.


213413_at
0.000589
−2.4
SBLF
stoned B-like factor (SBLF),
2p16.3






mRNA.


213418_at
3.79E−05
6.8
HSPA6
heat shock 70 kDa protein 6
1q23






(HSP70B′) (HSPA6), mRNA.


213464_at
0.0005718
2.7
SHC2
SHC (Src homology 2 domain
19p13.3






containing) transforming protein 2


213479_at
2.75E−05
4.3
NPTX2
neuronal pentraxin II (NPTX2),
7q21.3-q22.1






mRNA.


213523_at
5.86E−05
2.6
CCNE1
cyclin E1 (CCNE1), transcript
19q12






variant 2, mRNA.


213560_at
0.0005733
2.4
GADD45B
growth arrest and DNA-damage-
19p13.3






inducible, beta (GADD45B),






mRNA.


213574_s_at
1.00E−06
−2.4
KPNB1
Karyopherin (importin) beta 1
17q21.32


213605_s_at
0.0009961
2.5
FLJ40092
FLJ40092 protein
5q13.2


213661_at
8.00E−07
6.2
DKFZP586H2123
regeneration associated muscle
11p13






protease (DKFZP586H2123),






transcript variant 2, mRNA.


213687_s_at
0.0009746
−2.1
RPL35A
ribosomal protein L35a
3q29-qter






(RPL35A), mRNA.


213693_s_at
7.45E−05
6.2
MUC1
mucin 1, transmembrane
1q21






(MUC1), transcript variant 4,






mRNA.


213778_x_at
0.000467
2.2
ZFP276
zinc finger protein 276 homolog
16q24.3






(mouse) (ZFP276), mRNA.


213790_at
7.51E−05
4.4
ADAM12
A disintegrin and
10q26.3






metalloproteinase domain 12






(meltrin alpha)


213803_at
p < 1e−07
−3.6
KPNB1
Karyopherin (importin) beta 1
17q21.32


213836_s_at
0.0001261
2.2
WIPI49
WD40 repeat protein Interacting
17q24.2






with phosphoInositides of






49 kDa (WIPI49), mRNA.


213848_at
2.00E−07
3.1
DUSP7
Dual specificity phosphatase 7
3p21


213869_x_at
0.0002103
4.6
THY1
Thy-1 cell surface antigen
11q22.3-q23






(THY1), mRNA.


213895_at
0.0004485
−2.5
EMP1
Epithelial membrane protein 1
12p12.3


213900_at
3.89E−05
−2.7
C9orf61
chromosome 9 open reading
9q13-q21






frame 61 (C9orf61), mRNA.


213905_x_at
0.0001976
4.6
BGN
Biglycan
Xq28


213943_at
4.00E−07
19
TWIST1
twist homolog 1
7p21.2






(acrocephalosyndactyly 3;






Saethre-Chotzen syndrome)






(Drosophila) (TWIST1),






mRNA.


213979_s_at
0.0002226
3.3
CTBP1
C-terminal binding protein 1
4p16






(CTBP1), transcript variant 1,






mRNA.


214023_x_at
0.000646
2.8
MGC8685
Tubulin, beta polypeptide
6p25






paralog


214041_x_at
0.000362
2.8
RPL37A
Ribosomal protein L37a
2q35


214052_x_at
0.0005361
2.7
BAT2D1
BAT2 domain containing 1
1q23.3






(BAT2D1), mRNA.


214057_at
0.0005414
2.3
MCL1
myeloid cell leukemia sequence
1q21






1 (BCL2-related) (MCL1),






transcript variant 2, mRNA.


214081_at
4.00E−07
10.2
PLXDC1
plexin domain containing 1
17q21.1






(PLXDC1), mRNA.


214097_at
2.29E−05
−2.3
RPS21
Ribosomal protein S21
20q13.3


214110_s_at
0.0001953
3

ESTs, Highly similar to A43542






lymphocyte-specific protein 1






[H. sapiens]


214140_at
0.0004058
2
SLC25A16
solute carrier family 25
10q21.3






(mitochondrial carrier; Graves






disease autoantigen), member 16






(SLC25A16), nuclear gene






encoding mitochondrial protein,






mRNA.


214149_s_at
0.0003428
3.4
ATP6V0E
ATPase, H+ transporting,
5q35.1






lysosomal 9 kDa, V0 subunit e


214177_s_at
0.0001027
−2.2
PBXIP1
pre-B-cell leukemia
1q22






transcription factor interacting






protein 1 (PBXIP1), mRNA.


214264_s_at
0.0002244
2.1
C14orf143
chromosome 14 open reading
14q32.11






frame 143 (C14orf143), mRNA.


214316_x_at
7.97E−05
2.4
CALR
Calreticulin
19p13.3-p13.2


214359_s_at
0.0001495
−3
HSPCB
heat shock 90 kDa protein 1, beta
6p12






(HSPCB), mRNA.


214426_x_at
0.0008417
2.1
CHAF1A
chromatin assembly factor 1,
19p13.3






subunit A (p150) (CHAF1A),






mRNA.


214435_x_at
0.0005753
2.8
RALA
v-ral simian leukemia viral
7p15-p13






oncogene homolog A (ras






related) (RALA), mRNA.


214438_at
0.0009565
3.4
HLX1
H2.0-like homeo box 1
1q41-q42.1






(Drosophila) (HLX1), mRNA.


214527_s_at
4.20E−06
−2.3
PQBP1
polyglutamine binding protein 1
Xp11.23






(PQBP1), transcript variant 5,






mRNA.


214594_x_at
0.0002968
2.9
ATP8B1
ATPase, Class I, type 8B,
18q21-q22






member 1


214707_x_at
0.0003366
2.8
ALMS1
Alstrom syndrome 1
2p13


214715_x_at
0.0002241
4.3
ZNF160
zinc finger protein 160
19q13.41






(ZNF160), transcript variant 1,






mRNA.


214724_at
0.0006038
−2.1
DIXDC1
DIX domain containing 1
11q23.2






(DIXDC1), mRNA.


214737_x_at
0.0004018
−2.2
HNRPC
heterogeneous nuclear
14q11.2






ribonucleoprotein C (C1/C2)






(HNRPC), transcript variant 2,






mRNA.


214855_s_at
4.03E−05
−2.6
GARNL1
GTPase activating Rap/RanGAP
14q13.2






domain-like 1 (GARNL1),






transcript variant 2, mRNA.


214862_x_at
0.0009591
−2.8

MRNA; cDNA
10






DKFZp564G1162 (from clone






DKFZp564G1162)


214924_s_at
2.90E−06
3.8
OIP106
OGT(O-Glc-NAc transferase)-
3p25.3-p24.1






interacting protein 106 KDa






(OIP106), mRNA.


215016_x_at
1.50E−05
−2.3
DST
dystonin (DST), transcript
6p12-p11






variant 1, mRNA.


215046_at
0.0006908
−2.2
FLJ23861
hypothetical protein FLJ23861
2q34






(FLJ23861), mRNA.


215073_s_at
3.83E−05
−2.1
NR2F2
nuclear receptor subfamily 2,
15q26






group F, member 2 (NR2F2),






mRNA.


215076_s_at
0.000876
3
COL3A1
collagen, type III, alpha 1
2q31






(Ehlers-Danlos syndrome type






IV, autosomal dominant)






(COL3A1), mRNA.


215179_x_at
6.80E−05
4.5
PGF
Placental growth factor, vascular
14q24-q31






endothelial growth factor-related






protein


215203_at
0.0006265
2.2
GOLGA4
Golgi autoantigen, golgin
3p22-p21.3






subfamily a, 4


215206_at
0.0001618
3.1
EXT1
Exostoses (multiple) 1
8q24.11-q24.13


215208_x_at
0.0006292
3.4
RPL35A
Ribosomal protein L35a
3q29-qter


215294_s_at
8.91E−05
−2.9
SMARCA1
SWI/SNF related, matrix
Xq25






associated, actin dependent






regulator of chromatin,






subfamily a, member 1






(SMARCA1), transcript variant






2, mRNA.


215306_at
0.0008848
−3.4
LHCGR
Luteinizing
2p21






hormone/choriogonadotropin






receptor


215336_at
9.25E−05
2.7
AKAP11
A kinase (PRKA) anchor protein
13q14.11






11 (AKAP11), transcript variant






2, mRNA.


215373_x_at
0.0004506
2.8
SET8
PR/SET domain containing
12q24.31






protein 8


215383_x_at
0.0009763
2.6
SPG21
Spastic paraplegia 21
15q21-q22






(autosomal recessive, Mast






syndrome)


215404_x_at
0.0005104
3.2
FGFR1
Fibroblast growth factor
8p11.2-p11.1






receptor 1 (fms-related tyrosine






kinase 2, Pfeiffer syndrome)


215467_x_at
0.0004682
3.9
DHX9
DEAH (Asp-Glu-Ala-His) box
1q25






polypeptide 9


215504_x_at
0.0006038
2.5
ANKRD10
Ankyrin repeat domain 10
13q34


215529_x_at
0.0007444
2.6
C21orf106
Chromosome 21 open reading
21q22.3






frame 106


215566_x_at
0.0007954
2.2
LYPLA2
lysophospholipase II (LYPLA2),
1p36.12-p35.1






mRNA.


215577_at
5.29E−05
2.4
UBE2E1
Ubiquitin-conjugating enzyme
3p24.2






E2E 1 (UBC4/5 homolog, yeast)


215588_x_at
6.38E−05
3.7
RIOK3
RIO kinase 3 (yeast)
18q11.2


215599_at
4.10E−06
3.9
SMA4
SMA4
5q13


215600_x_at
0.0005929
3.3
FBXW12
F-box and WD-40 domain
3p21.31






protein 12


215604_x_at
0.0003643
3.8
UBE2D2
Ubiquitin-conjugating enzyme
5q31.2






E2D 2 (UBC4/5 homolog, yeast)


215628_x_at
0.0005005
2.7
PPP2CA
Protein phosphatase 2 (formerly
5q31.1






2A), catalytic subunit, alpha






isoform


215978_x_at
9.04E−05
5.6
LOC152719
ATP-binding cassette, sub-
4p16.3






family A (ABC1), member 11






(pseudogene)


216035_x_at
0.000632
−2
TCF7L2
Transcription factor 7-like 2 (T-
10q25.3






cell specific, HMG-box)


216051_x_at
5.83E−05
5
KIAA1217
KIAA1217
10p12.31


216187_x_at
0.0001456
3.7
XRCC3
X-ray repair complementing
14q32.3






defective repair in Chinese






hamster cells 3


216215_s_at
7.67E−05
−2.1
RBM9
RNA binding motif protein 9
22q13.1


216221_s_at
0.0007062
−2
PUM2
pumilio homolog 2 (Drosophila)
2p22-p21






(PUM2), mRNA.


216241_s_at
0.0005875
−2.1
TCEA1
transcription elongation factor A
8q11.2






(SII), 1 (TCEA1), transcript






variant 2, mRNA.


216246_at
0.0004251
2.7
RPS20
ribosomal protein S20 (RPS20),
8q12






mRNA.


216274_s_at
0.0003437
−2.3
SEC11L1
SEC11-like 1 (S. cerevisiae)
15q25.3






(SEC11L1), mRNA.


216733_s_at
0.0005519
−3.3
GATM
glycine amidinotransferase (L-
15q21.1






arginine:glycine






amidinotransferase) (GATM),






mRNA.


216858_x_at
0.0002005
4.1


216859_x_at
0.0004599
3.7


216944_s_at
0.0001923
−2.7
ITPR1
inositol 1,4,5-triphosphate
3p26-p25






receptor, type 1 (ITPR1),






mRNA.


217028_at
0.000384
3.4
CXCR4
chemokine (C—X—C motif)
2q21






receptor 4 (CXCR4), transcript






variant 2, mRNA.


217118_s_at
2.15E−05
2.2
C22orf9
chromosome 22 open reading
22q13.31






frame 9 (C22orf9), transcript






variant 2, mRNA.


217466_x_at
0.0009002
−2.2
RPS2
Ribosomal protein S2
16p13.3


217497_at
0.0002299
3.3
ECGF1
Endothelial cell growth factor 1
22q13






(platelet-derived)


217579_x_at
0.0006875
2.5
ARL6IP2
ADP-ribosylation factor-like 6
2p22.2-p22.1






interacting protein 2


217586_x_at
0.000568
2.9

ESTs


217679_x_at
7.86E−05
4.8

ESTs, Weakly similar to






hypothetical protein FLJ20489






[Homo sapiens] [H. sapiens]


217713_x_at
0.0001301
3.4

ESTs, Weakly similar to






ALU6_HUMAN ALU






SUBFAMILY SP SEQUENCE






CONTAMINATION






WARNING ENTRY






[H. sapiens]


217715_x_at
0.0002336
3.9

ESTs


217773_s_at
0.0002007
−2.4
NDUFA4
NADH dehydrogenase
7p21.3






(ubiquinone) 1 alpha






subcomplex, 4, 9 kDa






(NDUFA4), nuclear gene






encoding mitochondrial protein,






mRNA.


217774_s_at
0.0007057
−2.1
HSPC152
hypothetical protein HSPC152
11q13.1






(HSPC152), mRNA.


217781_s_at
0.0003457
−2.5
ZFP106
zinc finger protein 106 homolog
15q15.1






(mouse) (ZFP106), mRNA.


217787_s_at
8.07E−05
2.7
GALNT2
UDP-N-acetyl-alpha-D-
1q41-q42






galactosamine:polypeptide N-






acetylgalactosaminyltransferase






2 (GalNAc-T2) (GALNT2),






mRNA.


217795_s_at
0.0001341
−2.1
TMEM43
transmembrane protein 43
3p25.1






(TMEM43), mRNA.


217814_at
0.0004705
−2.2
GK001
GK001 protein (GK001),
17q23.3






mRNA.


217833_at
0.000595
−2.5
SYNCRIP
Synaptotagmin binding,
6q14-q15






cytoplasmic RNA interacting






protein


217862_at
0.000351
−2.6
PIAS1
Protein inhibitor of activated
15q






STAT, 1


217864_s_at
0.0004053
−2.2
PIAS1
protein inhibitor of activated
15q






STAT, 1 (PIAS1), mRNA.


217888_s_at
0.0003955
2.2
ARFGAP1
ADP-ribosylation factor GTPase
20q13.33






activating protein 1






(ARFGAP1), transcript variant






1, mRNA.


217915_s_at
9.25E−05
−2.2
C15orf15
chromosome 15 open reading
15q21






frame 15 (C15orf15), mRNA.


217989_at
2.94E−05
−2.7
DHRS8
dehydrogenase/reductase (SDR
4q22.1






family) member 8 (DHRS8),






mRNA.


217992_s_at
0.0003726
2.8
EFHD2
EF-hand domain family,
1p36.21






member D2 (EFHD2), mRNA.


218018_at
3.65E−05
2.2
PDXK
pyridoxal (pyridoxine, vitamin
21q22.3






B6) kinase (PDXK), mRNA.


218025_s_at
2.20E−06
−4.3
PECI
peroxisomal D3,D2-enoyl-CoA
6p24.3






isomerase (PECI), transcript






variant 1, mRNA.


218031_s_at
4.00E−07
−4.3
CHES1
checkpoint suppressor 1
14q24.3-q32.11






(CHES1), mRNA.


218113_at
0.000146
2
TMEM2
transmembrane protein 2
9q13-q21






(TMEM2), mRNA.


218130_at
0.0004049
2
MGC4368
hypothetical protein MGC4368
17q25.3






(MGC4368), mRNA.


218131_s_at
2.87E−05
2.9
GATAD2A
GATA zinc finger domain
19p13.11






containing 2A (GATAD2A),






mRNA.


218151_x_at
0.0005594
2.2
GPR172A
G protein-coupled receptor
8q24.3






172A (GPR172A), mRNA.


218155_x_at
0.000544
2.6
FLJ10534
hypothetical protein FLJ10534
17p13.3






(FLJ10534), mRNA.


218158_s_at
0.0001389
−2.7
APPL
adaptor protein containing pH
3p21.1-p14.3






domain, PTB domain and






leucine zipper motif 1 (APPL),






mRNA.


218167_at
7.25E−05
−2.4
AMZ2
archaemetzincins-2 (AMZ2),
17q24.2






mRNA.


218191_s_at
6.81E−05
−2.8
LMBRD1
LMBR1 domain containing 1
6q13






(LMBRD1), mRNA.


218193_s_at
0.0002316
2.8
GOLT1B
golgi transport 1 homolog B (S. cerevisiae)
12p12.1






(GOLT1B), mRNA.


218204_s_at
8.33E−05
−2.3
FYCO1
FYVE and coiled-coil domain
3p21.31






containing 1 (FYCO1), mRNA.


218311_at
0.0001916
−2.3
MAP4K3
mitogen-activated protein kinase
2p22.1






kinase kinase kinase 3






(MAP4K3), mRNA.


218373_at
0.0001922
−2.3
FTS
fused toes homolog (mouse)
16q12.2






(FTS), transcript variant 2,






mRNA.


218383_at
0.0003931
−2.7
C14orf94
chromosome 14 open reading
14q11.2






frame 94 (C14orf94), mRNA.


218432_at
0.0003175
−2.1
FBXO3
F-box protein 3 (FBXO3),
11p13






transcript variant 1, mRNA.


218450_at
0.0002984
−2.5
HEBP1
heme binding protein 1
12p13.1






(HEBP1), mRNA.


218504_at
0.0005544
−2
FAHD2A
fumarylacetoacetate hydrolase
2p24.3-p11.2






domain containing 2A






(FAHD2A), mRNA.


218528_s_at
4.23E−05
−2.3
RNF38
ring finger protein 38 (RNF38),
9p13-p12






transcript variant 4, mRNA.


218638_s_at
0.0008018
4
SPON2
spondin 2, extracellular matrix
4p16.3






protein (SPON2), mRNA.


218730_s_at
0.0002624
−6.8
OGN
osteoglycin (osteoinductive
9q22






factor, mimecan) (OGN),






transcript variant 3, mRNA.


218739_at
0.0002299
2.6
ABHD5
abhydrolase domain containing
3p21






5 (ABHD5), mRNA.


218804_at
1.58E−05
3.3
TMEM16A
transmembrane protein 16A
11q13.3






(TMEM16A), mRNA.


218817_at
0.0005521
2
SPCS3
signal peptidase complex
4q34.2






subunit 3 homolog (S. cerevisiae)






(SPCS3), mRNA.


218820_at
0.0007651
−3
C14orf132
chromosome 14 open reading
14q32.2






frame 132 (C14orf132), mRNA.


218831_s_at
7.99E−05
−2.6
FCGRT
Fc fragment of IgG, receptor,
19q13.3






transporter, alpha (FCGRT),






mRNA.


218856_at
2.20E−06
6.6
TNFRSF21
tumor necrosis factor receptor
6p21.1-12.2






superfamily, member 21






(TNFRSF21), mRNA.


218902_at
4.33E−05
2.5
NOTCH1
Notch homolog 1, translocation-
9q34.3






associated (Drosophila)






(NOTCH1), mRNA.


218919_at
0.0001794
−3.1
ZFAND1
zinc finger, AN1-type domain 1
8q21.13






(ZFAND1), mRNA.


218929_at
3.34E−05
−2.1
CARF
collaborates/cooperates with
4q35.1






ARF (alternate reading frame)






protein (CARF), mRNA.


218961_s_at
0.0007861
2
PNKP
polynucleotide kinase 3′-
19q13.3-q13.4






phosphatase (PNKP), mRNA.


219023_at
7.64E−05
−3.1
C4orf16
chromosome 4 open reading
4q25






frame 16 (C4orf16), mRNA.


219025_at
5.55E−05
4.3
CD248
CD248 antigen, endosialin
11q13






(CD248), mRNA.


219033_at
0.0007081
2.3
PARP8
poly (ADP-ribose) polymerase
5q11.1






family, member 8 (PARP8),






mRNA.


219054_at
4.17E−05
−2.6
FLJ14054
hypothetical protein FLJ14054
5p13.3






(FLJ14054), mRNA.


219092_s_at
2.14E−05
2.2
C9orf12
chromosome 9 open reading
9q21.33-q22.31






frame 12 (C9orf12), mRNA.


219099_at
9.73E−05
2
C12orf5
chromosome 12 open reading
12p13.3






frame 5 (C12orf5), mRNA.


219102_at
1.21E−05
3
RCN3
reticulocalbin 3, EF-hand
19q13.33






calcium binding domain






(RCN3), mRNA.


219105_x_at
9.00E−07
3.8
ORC6L
origin recognition complex,
16q12






subunit 6 homolog-like (yeast)






(ORC6L), mRNA.


219117_s_at
0.0002973
2.4
FKBP11
FK506 binding protein 11, 19 kDa
12q13.12






(FKBP11), mRNA.


219263_at
5.92E−05
4.2
RNF128
ring finger protein 128
Xq22.3






(RNF128), transcript variant 2,






mRNA.


219279_at
0.0002023
2.7
DOCK10
dedicator of cytokinesis 10
2q36.3






(DOCK10), mRNA.


219289_at
0.0001862
2.3
FLJ20718
hypothetical protein FLJ20718
16q12.1






(FLJ20718), transcript variant 1,






mRNA.


219290_x_at
0.0009817
3.8
DAPP1
dual adaptor of phosphotyrosine
4q25-q27






and 3-phosphoinositides






(DAPP1), mRNA.


219358_s_at
1.60E−06
6.1
CENTA2
centaurin, alpha 2 (CENTA2),
17q11.2






mRNA.


219359_at
4.00E−06
4.4
FLJ22635
hypothetical protein FLJ22635
11p15.5






(FLJ22635), mRNA.


219368_at
0.0009624
−2.9
NAP1L2
nucleosome assembly protein 1-
Xq13






like 2 (NAP1L2), mRNA.


219392_x_at
3.14E−05
4.3
FLJ11029
hypothetical protein FLJ11029
17q23.2






(FLJ11029), mRNA.


219407_s_at
0.0009809
4.1
LAMC3
laminin, gamma 3 (LAMC3),
9q31-q34






mRNA.


219449_s_at
0.0006528
−2.5
TMEM70
transmembrane protein 70
8q21.11






(TMEM70), mRNA.


219454_at
4.00E−07
36.8
EGFL6
EGF-like-domain, multiple 6
Xp22






(EGFL6), mRNA.


219493_at
0.0004597
2.6
SHCBP1
SHC SH2-domain binding
16q11.2






protein 1 (SHCBP1), mRNA.


219511_s_at
7.70E−06
−4.1
SNCAIP
synuclein, alpha interacting
5q23.1-q23.3






protein (synphilin) (SNCAIP),






mRNA.


219522_at
1.00E−07
7.7
FJX1
four jointed box 1 (Drosophila)
11p13






(FJX1), mRNA.


219549_s_at
0.0004199
−2.2
RTN3
reticulon 3 (RTN3), transcript
11q13






variant 4, mRNA.


219582_at
0.0004924
3
OGFRL1
opioid growth factor receptor-
6q13






like 1 (OGFRL1), mRNA.


219634_at
8.10E−06
3.8
CHST11
carbohydrate (chondroitin 4)
12q






sulfotransferase 11 (CHST11),






mRNA.


219641_at
0.0003308
−2.1
DET1
de-etiolated homolog 1
15q25.3






(Arabidopsis) (DET1), mRNA.


219700_at
1.10E−06
7.7
PLXDC1
plexin domain containing 1
17q21.1






(PLXDC1), mRNA.


219764_at
9.00E−07
6.7
FZD10
frizzled homolog 10
12q24.33






(Drosophila) (FZD10), mRNA.


219939_s_at
2.10E−06
−3.5
CSDE1
cold shock domain containing
1p22






E1, RNA-binding (CSDE1),






transcript variant 2, mRNA.


219958_at
0.000873
3.2
C20orf46
chromosome 20 open reading
20p13






frame 46 (C20orf46), mRNA.


219961_s_at
0.0003792
−2.5
C20orf19
chromosome 20 open reading
20pter-q11.23






frame 19 (C20orf19), mRNA.


220014_at
1.38E−05
4.6
LOC51334
mesenchymal stem cell protein
5q23.1






DSC54 (LOC51334), mRNA.


220094_s_at
0.0002242
2
C6orf79
chromosome 6 open reading
6p24.3-p23






frame 79 (C6orf79), transcript






variant 1, mRNA.


220113_x_at
0.0004047
3.1
POLR1B
polymerase (RNA) I polypeptide
2q13






B, 128 kDa (POLR1B), mRNA.


220167_s_at
0.0008187
2
TP53TG3
TP53TG3 protein (TP53TG3),
16p13






transcript variant 2, mRNA.


220232_at
4.54E−05
4.8
SCD5
stearoyl-CoA desaturase 5
4q21.3






(SCD5), mRNA.


220242_x_at
0.0001347
2.2
ZNF701
zinc finger protein 701
19q13.41






(ZNF701), mRNA.


220266_s_at
1.44E−05
−3.3
KLF4
Kruppel-like factor 4 (gut)
9q31






(KLF4), mRNA.


220301_at
1.76E−05
5.8
C18orf14
chromosome 18 open reading
18q22.1






frame 14 (C18orf14), mRNA.


220327_at
8.86E−05
−2.7
VGL-3
vestigial-like 3 (VGL-3),
3p12.1






mRNA.


220334_at
0.0003043
3.1
RGS17
regulator of G-protein signalling
6q25.3






17 (RGS17), mRNA.


220432_s_at
0.000473
−3.2
CYP39A1
cytochrome P450, family 39,
6p21.1-p11.2






subfamily A, polypeptide 1






(CYP39A1), mRNA.


220575_at
4.48E−05
3.4
FLJ11800
hypothetical protein FLJ11800
17p11.2






(FLJ11800), mRNA.


220603_s_at
0.0001617
3.9
MCTP2
Multiple C2-domains with two
15q26.2






transmembrane regions 2


220720_x_at
0.0005432
3.3
FLJ14346
Hypothetical protein FLJ14346
2q21.1


220796_x_at
0.0002356
4.1
SLC35E1
Solute carrier family 35,
19p13.11






member E1


220817_at
4.50E−06
4.1
TRPC4
transient receptor potential
13q13.1-q13.2






cation channel, subfamily C,






member 4 (TRPC4), mRNA.


220952_s_at
0.000293
−2.1
PLEKHA5
pleckstrin homology domain
12p12






containing, family A member 5






(PLEKHA5), mRNA.


220992_s_at
0.0003763
−2.1
C1orf25
Chromosome 1 open reading
1q25.2






frame 25


221012_s_at
0.0004784
2
TRIM8
tripartite motif-containing 8
10q24.3






(TRIM8), mRNA.


221059_s_at
2.20E−06
4.1
COTL1
coactosin-like 1 (Dictyostelium)
16q24.1






(COTL1), mRNA.


221127_s_at
0.000454
3.7
DKK3
dickkopf homolog 3 (Xenopus
11p15.2






laevis) (DKK3), transcript






variant 3, mRNA.


221222_s_at
0.0005338
2.2
C1orf56
chromosome 1 open reading
1q21.2






frame 56 (C1orf56), mRNA.


221476_s_at
4.74E−05
−2.4
RPL15
ribosomal protein L15 (RPL15),
3p24.2






mRNA.


221486_at
0.0004058
−2
ENSA
endosulfine alpha (ENSA),
1q21.2






transcript variant 7, mRNA.


221538_s_at
0.0004626
2.4
PLXNA1
plexin A1 (PLXNA1), mRNA.
3q21.3


221558_s_at
1.86E−05
4.6
LEF1
lymphoid enhancer-binding
4q23-q25






factor 1 (LEF1), mRNA.


221577_x_at
0.0006888
4.5
GDF15
growth differentiation factor 15
19p13.1-13.2






(GDF15), mRNA.


221588_x_at
0.0001441
−2.4
ALDH6A1
aldehyde dehydrogenase 6
14q24.3






family, member A1






(ALDH6A1), nuclear gene






encoding mitochondrial protein,






mRNA.


221589_s_at
1.20E−06
−4.3
ALDH6A1
Aldehyde dehydrogenase 6
14q24.3






family, member A1


221590_s_at
0.0008671
−2.3
ALDH6A1
Aldehyde dehydrogenase 6
14q24.3






family, member A1


221689_s_at
0.000155
−2
DSCR5
Down syndrome critical region
21q22.2






gene 5 (DSCR5), transcript






variant 2, mRNA.


221691_x_at
1.46E−05
−3.2
NPM1
nucleophosmin (nucleolar
5q35






phosphoprotein B23, numatrin)






(NPM1), mRNA.


221725_at
3.60E−05
−2.1
WASF2
WAS protein family, member 2
1p36.11-p34.3


221726_at
1.93E−05
−2.9
RPL22
ribosomal protein L22 (RPL22),
1p36.3-p36.2






mRNA.


221727_at
0.0001534
−2.3
PC4
Activated RNA polymerase II
5p13.3






transcription cofactor 4


221729_at
0.0004594
3.1
COL5A2
collagen, type V, alpha 2
2q14-q32






(COL5A2), mRNA.


221730_at
0.0007972
3.4
COL5A2
collagen, type V, alpha 2
2q14-q32






(COL5A2), mRNA.


221731_x_at
2.57E−05
10.4
CSPG2
chondroitin sulfate proteoglycan
5q14.3






2 (versican) (CSPG2), mRNA.


221747_at
0.0003412
−2.2
TNS
Tensin 1
2q35-q36


221771_s_at
0.000498
−2.1
HSMPP8
M-phase phosphoprotein, mpp8
13q12.11


221840_at
0.0002221
3.2
PTPRE
protein tyrosine phosphatase,
10q26






receptor type, E (PTPRE),






transcript variant 2, mRNA.


221882_s_at
0.0003013
2.2
TMEM8
transmembrane protein 8 (five
16p13.3






membrane-spanning domains)






(TMEM8), mRNA.


221943_x_at
0.0002266
2.4
RPL38
ribosomal protein L38 (RPL38),
17q23-q25






mRNA.


221988_at
1.59E−05
−2.3
MGC2747
Hypothetical protein MGC2747
19p13.11


222108_at
5.57E−05
−4.4
AMIGO2
adhesion molecule with Ig-like
12q13.11






domain 2 (AMIGO2), mRNA.


222207_x_at
0.0003362
3.2

CDNA: FLJ20949 fis, clone
7






ADSE01902


222212_s_at
0.0001439
−2.3
LASS2
LAG1 longevity assurance
1q21.2






homolog 2 (S. cerevisiae)






(LASS2), transcript variant 3,






mRNA.


222252_x_at
5.29E−05
4
LRRC51
leucine rich repeat containing 51
11q13.4






(LRRC51), mRNA.


222253_s_at
0.000969
4.6
DKFZP434P211
POM121-like protein
22q11.22


222358_x_at
7.05E−05
3.1

ESTs, Weakly similar to






hypothetical protein FLJ20378






[Homo sapiens] [H. sapiens]


222372_at
0.0006654
2.5
BAIAP1
Membrane associated guanylate
3p14.1






kinase, WW and PDZ domain






containing 1


222379_at
1.13E−05
3.5
KCNE4
Potassium voltage-gated
2q36.3






channel, Isk-related family,






member 4


222394_at
1.44E−05
−2.8
PDCD6IP
programmed cell death 6
3p23






interacting protein (PDCD6IP),






mRNA.


222423_at
9.81E−05
−2.8
NDFIP1
Nedd4 family interacting protein 1
5q31.3


222431_at
0.0002108
−2.2
SPIN
Spindlin
9q22.1-q22.3


222437_s_at
0.0006141
−2
VPS24
vacuolar protein sorting 24
2p24.3-p24.1






(yeast) (VPS24), transcript






variant 2, mRNA.


222449_at
7.80E−06
4.1
TMEPAI
transmembrane, prostate
20q13.31-q13.33






androgen induced RNA






(TMEPAI), transcript variant 3,






mRNA.


222453_at
4.19E−05
−2.7
CYBRD1
cytochrome b reductase 1
2q31.1






(CYBRD1), mRNA.


222482_at
1.85E−05
−2.4
SSBP3
Single stranded DNA binding
1p32.3






protein 3


222486_s_at
5.80E−06
−4.9
ADAMTS1
ADAM metallopeptidase with
21q21.2






thrombospondin type 1 motif, 1






(ADAMTS1), mRNA.


222488_s_at
9.10E−05
−2.4
DCTN4
dynactin 4 (p62) (DCTN4),
5q31-q32






mRNA.


222494_at
4.00E−06
−2.8
CHES1
checkpoint suppressor 1
14q24.3-q32.11






(CHES1), mRNA.


222503_s_at
0.0003949
2.5
WDR41
WD repeat domain 41
5q13.3






(WDR41), mRNA.


222533_at
0.0001299
−2.1
CRBN
cereblon (CRBN), mRNA.
3p26.2


222538_s_at
0.0007304
−3.2
APPL
adaptor protein containing pH
3p21.1-p14.3






domain, PTB domain and






leucine zipper motif 1 (APPL),






mRNA.


222605_at
1.64E−05
−3.1
RCOR3
REST corepressor 3 (RCOR3),
1q32.3






mRNA.


222722_at
1.25E−05
−8.7
OGN
osteoglycin (osteoinductive
9q22






factor, mimecan) (OGN),






transcript variant 3, mRNA.


222753_s_at
0.0001335
2.1
SPCS3
signal peptidase complex
4q34.2






subunit 3 homolog (S. cerevisiae)






(SPCS3), mRNA.


222791_at
1.20E−06
−3.4
RSBN1
round spermatid basic protein 1
1p13.2






(RSBN1), mRNA.


222834_s_at
0.0006523
−2.1
GNG12
guanine nucleotide binding
1p31.2






protein (G protein), gamma 12






(GNG12), mRNA.


222968_at
5.98E−05
2.9
C6orf48
chromosome 6 open reading
6p21.3






frame 48 (C6orf48), mRNA.


222975_s_at
9.41E−05
−2.9
CSDE1
cold shock domain containing
1p22






E1, RNA-binding (CSDE1),






transcript variant 2, mRNA.


223007_s_at
0.0007305
−2.7
C9orf5
chromosome 9 open reading
9q31






frame 5 (C9orf5), mRNA.


223010_s_at
0.0005426
−2.2
OCIAD1
OCIA domain containing 1
4p11






(OCIAD1), mRNA.


223011_s_at
0.0008498
−2
OCIAD1
OCIA domain containing 1
4p11






(OCIAD1), mRNA.


223050_s_at
0.0003276
2
FBXW5
F-box and WD-40 domain
9q34.3






protein 5 (FBXW5), transcript






variant 1, mRNA.


223082_at
0.0003668
2.1
SH3KBP1
SH3-domain kinase binding
Xp22.1-p21.3






protein 1 (SH3KBP1), transcript






variant 1, mRNA.


223170_at
0.0001428
−2.6
DKFZP564K1964
DKFZP564K1964 protein
17q11.2






(DKFZP564K1964), mRNA.


223189_x_at
9.46E−05
−2.4
MLL5
myeloid/lymphoid or mixed-
7q22.1






lineage leukemia 5 (trithorax






homolog, Drosophila) (MLL5),






mRNA.


223208_at
0.0009547
2
KCTD10
potassium channel
12q24.11






tetramerisation domain






containing 10 (KCTD10),






mRNA.


223227_at
0.0006403
−2.2
BBS2
Bardet-Biedl syndrome 2
16q21






(BBS2), mRNA.


223263_s_at
0.0003743
−2.1
FGFR1OP2
FGFR1 oncogene partner 2
12p11.23






(FGFR1OP2), mRNA.


223276_at
5.10E−05
2.9
NID67
putative small membrane protein
5q33.1






NID67 (NID67), mRNA.


223283_s_at
0.0009337
−2.5
SDCCAG33
serologically defined colon
18q22.3






cancer antigen 33






(SDCCAG33), mRNA.


223306_at
6.02E−05
−2.4
EBPL
emopamil binding protein-like
13q12-q13






(EBPL), mRNA.


223366_at
0.0008475
−2.9

CDNA FLJ16218 fis, clone
8






CTONG3001501, highly similar






to Mus musculus glucocorticoid-






induced gene 1 mRNA


223384_s_at
0.0009265
−2.1
TRIM4
tripartite motif-containing 4
7q22-q31.1






(TRIM4), transcript variant beta,






mRNA.


223395_at
9.35E−05
−6.6
ABI3BP
ABI gene family, member 3
3q12






(NESH) binding protein






(ABI3BP), mRNA.


223437_at
0.0004419
−2.2
PPARA
peroxisome proliferative
22q13.31






activated receptor, alpha






(PPARA), transcript variant 3,






mRNA.


223464_at
0.0004952
2.3
OSBPL5
oxysterol binding protein-like 5
11p15.4






(OSBPL5), transcript variant 2,






mRNA.


223501_at
0.0004401
4.3
TNFSF13B
Tumor necrosis factor (ligand)
13q32-34






superfamily, member 13b


223538_at
0.0008098
2
SERF1A
Small EDRK-rich factor 1A
5q12.2-q13.3






(telomeric)


223566_s_at
0.0007174
−2.7
BCOR
BCL6 co-repressor (BCOR),
Xp21.2-p11.4






transcript variant 1, mRNA.


223617_x_at
3.01E−05
2.6
ATAD3B
ATPase family, AAA domain
1p36.33






containing 3B (ATAD3B),






mRNA.


223629_at
0.0008805
−2.4
PCDHB5
protocadherin beta 5
5q31






(PCDHB5), mRNA.


223672_at
0.0001002
4.8
SGIP1
SH3-domain GRB2-like
1p31.2






(endophilin) interacting protein






1 (SGIP1), mRNA.


223697_x_at
0.0001465
4.6
C9orf64
chromosome 9 open reading
9q21.32






frame 64 (C9orf64), mRNA.


223991_s_at
2.42E−05
2.6
GALNT2
UDP-N-acetyl-alpha-D-
1q41-q42






galactosamine:polypeptide N-






acetylgalactosaminyltransferase






2 (GalNAc-T2) (GALNT2),






mRNA.


224254_x_at
2.71E−05
5.5
TF
Transferrin
3q22.1


224445_s_at
1.11E−05
−3
ZFYVE21
zinc finger, FYVE domain
14q32.33






containing 21 (ZFYVE21),






mRNA.


224549_x_at
6.50E−06
8


224598_at
3.68E−05
2.4
MGAT4B
mannosyl (alpha-1,3-)-
5q35






glycoprotein beta-1,4-N-






acetylglucosaminyltransferase,






isoenzyme B (MGAT4B),






transcript variant 1, mRNA.


224605_at
0.0002548
−2.2
LOC401152
HCV F-transactivated protein 1
4q26






(LOC401152), mRNA.


224612_s_at
6.79E−05
2.2
DNAJC5
DnaJ (Hsp40) homolog,
20q13.33






subfamily C, member 5


224618_at
0.0001475
2
ROD1
ROD1 regulator of
9q32






differentiation 1 (S. pombe)


224660_at
0.0008567
−2.1
MGC14156
hypothetical protein MGC14156
4q22.1






(MGC14156), mRNA.


224664_at
0.0003695
−2.3
C10orf104
chromosome 10 open reading
10q22.1






frame 104 (C10orf104), mRNA.


224665_at
0.0004075
−2.2
C10orf104
chromosome 10 open reading
10q22.1






frame 104 (C10orf104), mRNA.


224667_x_at
0.0002844
3.1

Transcribed locus


224689_at
0.0001149
−2
MANBAL
mannosidase, beta A, lysosomal-
20q11.23-q12






like (MANBAL), transcript






variant 2, mRNA.


224734_at
0.0001511
−2.9
HMGB1
High-mobility group box 1
13q12


224741_x_at
0.0001144
−2.5
GAS5
Growth arrest-specific 5
1q23.3


224754_at
0.000206
−2.2
SP1
Sp1 transcription factor (SP1),
12q13.1






mRNA.


224755_at
0.0001441
−2.2
SMBP
SM-11044 binding protein
10q24.1


224763_at
5.30E−06
−3.9
RPL37
ribosomal protein L37 (RPL37),
5p13






mRNA.


224780_at
0.0008827
−2
RBM17
RNA binding motif protein 17
10p15.1






(RBM17), mRNA.


224812_at
0.0001992
−2.3
HIBADH
3-hydroxyisobutyrate
7p15.2






dehydrogenase (HIBADH),






mRNA.


224841_x_at
0.0001078
−2.6
RNU47
PREDICTED: RNA, U47 small
1






nuclear (RNU47), misc RNA.


224856_at
0.0004439
−2.6
FKBP5
FK506 binding protein 5
6p21.3-21.2






(FKBP5), mRNA.


224893_at
0.0003585
−2.8
DKFZP564J0863
DKFZP564J0863 protein
11q13.1


224895_at
0.000307
−2.6
YAP1
Yes-associated protein 1, 65 kDa
11q13






(YAP1), mRNA.


224901_at
1.70E−05
−3.5
SCD4
Stearoyl-CoA desaturase 5
4q21.3


224950_at
0.0009862
2.2
PTGFRN
prostaglandin F2 receptor
1p13.1






negative regulator (PTGFRN),






mRNA.


224967_at
5.98E−05
2.7
UGCG
UDP-glucose ceramide
9q31






glucosyltransferase


224970_at
5.88E−05
−2.5
NFIA
Nuclear factor I/A
1p31.3-p31.2


225050_at
4.85E−05
−2.5
ZNF512
zinc finger protein 512
2p23






(ZNF512), mRNA.


225060_at
0.0001496
−2.4
LRP11
low density lipoprotein receptor-
6q25.1






related protein 11 (LRP11),






mRNA.


225078_at
0.0002343
−3.4
EMP2
Epithelial membrane protein 2
16p13.2


225098_at
0.0001021
−2
ABI2
Abl interactor 2
2q33


225106_s_at
0.0005784
2.9
FLJ10826
hypothetical protein FLJ10826
16q12.2






(FLJ10826), transcript variant 2,






mRNA.


225123_at
4.00E−07
−3.1
SESN3
Sestrin 3
11q21


225125_at
3.50E−05
−3.1
TMEM32
transmembrane protein 32
Xq26.3






(TMEM32), mRNA.


225132_at
0.0001306
−2
FBXL3
F-box and leucine-rich repeat
13q22






protein 3 (FBXL3), mRNA.


225133_at
4.79E−05
−2.6
KLF3
Kruppel-like factor 3 (basic)
4p14


225147_at
1.50E−06
3.7
PSCD3
pleckstrin homology, Sec7 and
7p22.1






coiled-coil domains 3 (PSCD3),






mRNA.


225162_at
1.40E−05
−3.8
SH3D19
SH3 domain protein D19
4q31.3






(SH3D19), mRNA.


225179_at
0.0001313
−2.1
HIP2
Huntingtin interacting protein 2
4p14


225198_at
2.56E−05
−2.7
VAPA
VAMP (vesicle-associated
18p11.22






membrane protein)-associated






protein A, 33 kDa (VAPA),






transcript variant 2, mRNA.


225207_at
0.0001018
−3.4
PDK4
pyruvate dehydrogenase kinase,
7q21.3-q22.1






isoenzyme 4 (PDK4), mRNA.


225219_at
2.70E−05
−3.5
SMAD5
SMAD, mothers against DPP
5q31






homolog 5 (Drosophila)






(SMAD5), transcript variant 3,






mRNA.


225220_at
0.00074
−2.2

Homo sapiens, clone
4






IMAGE: 4249217, mRNA


225223_at
2.89E−05
−2
SMAD5
SMAD, mothers against DPP
5q31






homolog 5 (Drosophila)






(SMAD5), transcript variant 3,






mRNA.


225239_at
0.0004432
4.3

Immunoglobulin light chain
11






variable region


225243_s_at
0.0001227
−2.6
SLMAP
sarcolemma associated protein
3p21.2-p14.3






(SLMAP), mRNA.


225274_at
2.80E−06
−3
SNRPG
Small nuclear ribonucleoprotein
2p13.3






polypeptide G


225310_at
0.000177
−2.2
RBMX
RNA binding motif protein, X-
Xq26.3






linked


225326_at
8.88E−05
−2
RBM27
PREDICTED: RNA binding
5






motif protein 27 (RBM27),






mRNA.


225330_at
0.0002697
−2.1
IGF1R
Insulin-like growth factor 1
15q26.3






receptor


225332_at
5.78E−05
−2.1
KRTAP4-7
Keratin associated protein 4-7
17q12-q21


225344_at
0.0004132
2.8
NCOA7
nuclear receptor coactivator 7
6q22.32






(NCOA7), mRNA.


225352_at
0.0001828
−2.3
TLOC1
translocation protein 1
3q26.2






(TLOC1), mRNA.


225381_at
0.0006635
−4.3
LOC399959
PREDICTED: hypothetical
11






LOC399959 (LOC399959),






mRNA.


225387_at
3.43E−05
−3.2
TM4SF9
Tetraspanin 5
4q23


225416_at
0.0003496
−2.2
RNF12
Ring finger protein 12
Xq13-q21


225421_at
0.0009226
−3.1
ACY1L2
aminoacylase 1-like 2
6q15






(ACY1L2), mRNA.


225426_at
6.78E−05
−2.5
PPP6C
Protein phosphatase 6, catalytic
9q33.3






subunit


225480_at
0.0002718
2.2
C1orf122
chromosome 1 open reading
1p34.3






frame 122 (C1orf122), mRNA.


225489_at
3.71E−05
−2
TMEM18
transmembrane protein 18
2p25.3






(TMEM18), mRNA.


225498_at
7.45E−05
−2.2
CHMP4B
chromatin modifying protein 4B
20q11.22






(CHMP4B), mRNA.


225505_s_at
1.90E−06
4.1
C20orf81
chromosome 20 open reading
20p13






frame 81 (C20orf81), mRNA.


225509_at
1.01E−05
−2.9
SAP30L
Hypothetical protein LOC56757
5q33.2


225524_at
5.00E−05
−3.1
ANTXR2
anthrax toxin receptor 2
4q21.21






(ANTXR2), mRNA.


225526_at
0.0004149
−2
MKLN1
muskelin 1, intracellular
7q32






mediator containing kelch






motifs (MKLN1), mRNA.


225546_at
4.45E−05
−2.7
EEF2K
Eukaryotic elongation factor-2
16p12.1






kinase


225571_at
0.000188
−4.7
LIFR
leukemia inhibitory factor
5p13-p12






receptor (LIFR), mRNA.


225574_at
8.57E−05
−2.4
MGC10198
hypothetical protein MGC10198
4q35.1






(MGC10198), mRNA.


225575_at
0.0003237
−4.5
LIFR
leukemia inhibitory factor
5p13-p12






receptor (LIFR), mRNA.


225611_at
0.0004119
−2.6
MAST4
Microtubule associated
5q12.3






serine/threonine kinase family






member 4


225626_at
0.0003153
4.8
PAG1
phosphoprotein associated with
8q21.13






glycosphingolipid microdomains






1 (PAG1), mRNA.


225636_at
0.000278
2.5
STAT2
signal transducer and activator
12q13.3






of transcription 2, 113 kDa






(STAT2), mRNA.


225646_at
1.50E−05
4.4
CTSC
cathepsin C (CTSC), transcript
11q14.1-q14.3






variant 2, mRNA.


225647_s_at
0.0009077
2.5
CTSC
cathepsin C (CTSC), transcript
11q14.1-q14.3






variant 1, mRNA.


225686_at
0.0003333
2.2
FAM33A
family with sequence similarity
17q23.2






33, member A (FAM33A),






mRNA.


225698_at
3.41E−05
−3.2
TIGA1
TIGA1 (TIGA1), mRNA.
5q21-q22


225728_at
0.0006083
−2.8
ARGBP2
Arg/Abl-interacting protein
4q35.1






ArgBP2


225793_at
0.000103
−2.1
MGC46719
Lix1 homolog (mouse) like
1q21.1


225799_at
2.29E−05
5.2
MGC4677
hypothetical protein MGC4677
2p11.2






(MGC4677), mRNA.


225811_at
5.44E−05
−2.1

Transcribed locus, weakly
11






similar to XP_510104.1






PREDICTED: similar to






hypothetical protein FLJ25224






[Pan troglodytes]


225845_at
0.0003774
−3.4
BTBD15
BTB (POZ) domain containing
11q24.3






15 (BTBD15), mRNA.


225855_at
7.78E−05
−2.4
EPB41L5
erythrocyte membrane protein
2q14.2






band 4.1 like 5 (EPB41L5),






mRNA.


225886_at
0.0005086
−2.2
DDX5
RNA-binding protein 45
17q21






(RBP45), putative


225915_at
3.50E−05
−3.4
CAB39L
Calcium binding protein 39-like
13q14.2


225939_at
0.0001338
−2.8
EIF4E3
Eukaryotic translation initiation
3p14






factor 4E member 3


225941_at
0.0002862
−2.2
EIF4E3
Eukaryotic translation initiation
3p14






factor 4E member 3


225946_at
0.0003511
−2.3
C12orf2
Chromosome 12 open reading
12p12.3






frame 2


225947_at
5.75E−05
2.3
MYOHD1
myosin head domain containing
17q12






1 (MYOHD1), mRNA.


225967_s_at
0.0001375
2.5
LOC284184
PREDICTED: hypothetical
17






LOC284184 (LOC284184),






mRNA.


225976_at
0.0003271
−2
BTF3L4
basic transcription factor 3-like
1p32.3






4 (BTF3L4), mRNA.


225987_at
0.0003503
3.3
TNFAIP9
STEAP family member 4
7q21.12


225996_at
0.0006306
−9.3

MRNA; cDNA
2






DKFZp686N1345 (from clone






DKFZp686N1345)


226017_at
0.000301
2.3
CKLFSF7
chemokine-like factor
3p23






superfamily 7 (CKLFSF7),






transcript variant 2, mRNA.


226020_s_at
7.89E−05
−2.2
OMA1
OMA1 homolog, zinc
1p32.2-p32.1






metallopeptidase (S. cerevisiae)






(OMA1), mRNA.


226038_at
0.0002389
−3.2
LONRF1
LON peptidase N-terminal
8p23.1






domain and ring finger 1






(LONRF1), mRNA.


226063_at
4.00E−06
3.3
VAV2
vav 2 oncogene (VAV2),
9q34.1






mRNA.


226066_at
0.0005179
−3
MITF
microphthalmia-associated
3p14.2-p14.1






transcription factor (MITF),






transcript variant 5, mRNA.


226117_at
8.72E−05
−2.7
TIFA
TRAF-interacting protein with a
4q25






forkhead-associated domain






(TIFA), mRNA.


226120_at
0.0001436
−2.7
TTC8
tetratricopeptide repeat domain
14q31.3






8 (TTC8), transcript variant 3,






mRNA.


226180_at
0.0009691
−2
WDR36
WD repeat domain 36
5q22.1






(WDR36), mRNA.


226184_at
3.75E−05
−3.6
FMNL2
formin-like 2 (FMNL2),
2q23.3






transcript variant 2, mRNA.


226203_at
0.0003968
−2

CDNA clone IMAGE: 5299888
15


226223_at
0.0002488
−2.6
PAWR
PRKC, apoptosis, WT1,
12q21






regulator


226225_at
0.0003786
−3.2
MCC
Mutated in colorectal cancers
5q21-q22


226230_at
0.0002589
−2.4
KIAA1387
KIAA1387 protein
2p16.1


226280_at
3.34E−05
−2.8
BNIP2
BCL2/adenovirus E1B 19 kDa
15q22.2






interacting protein 2


226297_at
8.43E−05
−2.1

ESTs


226303_at
0.0003656
−2.9
PGM5
phosphoglucomutase 5 (PGM5),
9q13






mRNA.


226336_at
0.0001456
−2
PPIA
Peptidylprolyl isomerase A
7p13-p11.2






(cyclophilin A)


226344_at
0.0009627
−2.9
ZMAT1
zinc finger, matrin type 1
Xq21






(ZMAT1), transcript variant 1,






mRNA.


226403_at
9.20E−06
3.8
TMC4
transmembrane channel-like 4
19q13.42






(TMC4), mRNA.


226472_at
0.0007652
−2
PPIL4
peptidylprolyl isomerase
6q24-q25






(cyclophilin)-like 4 (PPIL4),






mRNA.


226484_at
0.0009077
2
ZNF651
zinc finger protein 651
3p22.1






(ZNF651), mRNA.


226499_at
0.0009453
2.8
MGC61598
Similar to ankyrin-repeat protein
9q34.3






Nrarp


226521_s_at
0.0006084
−2.1
FLJ13614
hypothetical protein FLJ13614
4q21.21-q21.23






(FLJ13614), mRNA.


226529_at
4.85E−05
−2.3
FLJ11273
hypothetical protein FLJ11273
7p21.3






(FLJ11273), mRNA.


226541_at
0.0001172
−2.3
FBXO30
F-box protein 30 (FBXO30),
6q24






mRNA.


226561_at
0.0006025
−2.1
LOC285086
Hypothetical protein
2q36.3






LOC285086


226599_at
0.0002151
2.3
KIAA1727
KIAA1727 protein
4q31.3






(KIAA1727), mRNA.


226625_at
7.30E−06
−3.8
TGFBR3
Transforming growth factor,
1p33-p32






beta receptor III (betaglycan,






300 kDa)


226663_at
8.60E−06
2.8
ANKRD10
Ankyrin repeat domain 10
13q34


226668_at
0.0003707
−2.6
WDSUB1
WD repeat, SAM and U-box
2q24.2






domain containing 1






(WDSUB1), mRNA.


226688_at
0.0002101
−3.3
C3orf23
chromosome 3 open reading
3p21.33-p21.32






frame 23 (C3orf23), transcript






variant 1, mRNA.


226695_at
0.0001301
2.5
PRRX1
paired related homeobox 1
1q24






(PRRX1), transcript variant






pmx-1b, mRNA.


226705_at
0.0002111
−2
FGFR1
Fibroblast growth factor
8p11.2-p11.1






receptor 1 (fms-related tyrosine






kinase 2, Pfeiffer syndrome)


226713_at
1.81E−05
−2.9
C3orf6
Chromosome 3 open reading
3






frame 6


226747_at
0.0006894
−3.7
KIAA1344
KIAA1344 (KIAA1344),
14q22.1






mRNA.


226751_at
8.30E−06
−2.4
C2orf32
chromosome 2 open reading
2p14






frame 32 (C2orf32), mRNA.


226765_at
0.0002842
2.2
SPTBN1
Spectrin, beta, non-erythrocytic 1
2p21


226777_at
5.39E−05
4.9
ADAM12
A disintegrin and
10q26.3






metalloproteinase domain 12






(meltrin alpha)


226806_s_at
1.95E−05
−2.8

MRNA; cDNA
1






DKFZp686J23256 (from clone






DKFZp686J23256)


226829_at
0.0006675
2.9
KIAA1914
KIAA1914 (KIAA1914),
10q25.3






transcript variant 2, mRNA.


226867_at
0.0008554
−2
C9orf55
Chromosome 9 open reading
9p22.1






frame 55


226873_at
0.0002067
−2.8

Transcribed locus
16


226899_at
3.10E−06
3.1
UNC5B
unc-5 homolog B (C. elegans)
10q22.1






(UNC5B), mRNA.


226909_at
0.0004528
−2
KIAA1729
KIAA1729 protein
4p16.1






(KIAA1729), mRNA.


226911_at
1.20E−05
3.7
FLJ39155
hypothetical protein FLJ39155
5p13.2-p13.1






(FLJ39155), transcript variant 4,






mRNA.


226933_s_at
5.79E−05
3.8
ID4
inhibitor of DNA binding 4,
6p22-p21






dominant negative helix-loop-






helix protein (ID4), mRNA.


226943_at
0.0004664
2

MRNA; cDNA DKFZp547P055
12






(from clone DKFZp547P055)


226994_at
0.0002644
−2.3
DNAJA2
DnaJ (Hsp40) homolog,
16q11.1-q11.2






subfamily A, member 2


226997_at
0.0002372
3.7

CDNA FLJ10196 fis, clone
5






HEMBA1004776


227031_at
0.0001825
−2.4
SNX13
Sorting nexin 13
7p21.1


227070_at
0.0003008
−2.7
GLT8D2
glycosyltransferase 8 domain
12q






containing 2 (GLT8D2), mRNA.


227082_at
0.0007234
−2

MRNA; cDNA
3






DKFZp586K1922 (from clone






DKFZp586K1922)


227121_at
0.0006088
−2.1

MRNA; cDNA
3






DKFZp586K1922 (from clone






DKFZp586K1922)


227132_at
0.0002728
−2.4
LOC51123
HSPC038 protein (LOC51123),
8q22.3






mRNA.


227138_at
0.0008198
−2.1
CRTAP
cartilage associated protein
3p22.3






(CRTAP), mRNA.


227148_at
0.0001511
−3.4
PLEKHH2
pleckstrin homology domain
2p21






containing, family H (with






MyTH4 domain) member 2






(PLEKHH2), mRNA.


227178_at
0.0005124
−3.9
CUGBP2
CUG triplet repeat, RNA
10p13






binding protein 2 (CUGBP2),






transcript variant 2, mRNA.


227197_at
0.000108
−2.9
SGEF
Src homology 3 domain-
3q25.2






containing guanine nucleotide






exchange factor


227214_at
0.0002058
2.1
GOPC
Golgi associated PDZ and
6q21






coiled-coil motif containing


227221_at
0.0004345
2.1

CDNA FLJ31683 fis, clone
3






NT2RI2005353


227260_at
0.0001678
3

Transcribed locus
1


227273_at
4.47E−05
−2.6

Transcribed locus
10


227278_at
0.0005386
2.3

Transcribed locus, weakly
1






similar to XP_510104.1






PREDICTED: similar to






hypothetical protein FLJ25224






[Pan troglodytes]


227293_at
8.47E−05
−2.2
LNX
Ligand of numb-protein X
4q12


227295_at
2.70E−06
3.4
IKIP
IKK interacting protein (IKIP),
12q23.1






transcript variant 3.1, mRNA.


227317_at
0.0003721
2.3
LMCD1
LIM and cysteine-rich domains
3p26-p24






1 (LMCD1), mRNA.


227347_x_at
2.86E−05
4
HES4
hairy and enhancer of split 4
1p36.33






(Drosophila) (HES4), mRNA.


227372_s_at
0.0004102
3.4
BAIAP2L1
BAI1-associated protein 2-like 1
7q21.3-q22.1






(BAIAP2L1), mRNA.


227383_at
0.0005636
2.4

Similar to KIAA0454 protein
1q21.1


227384_s_at
0.000128
2.6

Similar to KIAA0454 protein
1q21.1


227396_at
0.0001476
2.6

Homo sapiens, clone
11






IMAGE: 4454331, mRNA


227407_at
9.42E−05
−2.3
FLJ90013
hypothetical protein FLJ90013
4p15.32






(FLJ90013), mRNA.


227529_s_at
0.0001731
−2.9
AKAP12
A kinase (PRKA) anchor protein
6q24-q25






(gravin) 12


227530_at
8.47E−05
−3
AKAP12
A kinase (PRKA) anchor protein
6q24-q25






(gravin) 12


227636_at
0.0003391
−2.3
THAP5
THAP domain containing 5
7q31.1


227703_s_at
1.29E−05
−5.9
SYTL4
synaptotagmin-like 4
Xq21.33






(granuphilin-a) (SYTL4),






mRNA.


227708_at
0.0006098
−2.1
EEF1A1
Eukaryotic translation
6q14.1






elongation factor 1 alpha 1


227719_at
4.00E−06
−3.6

CDNA FLJ37828 fis, clone
13






BRSSN2006575


227728_at
6.11E−05
−2.9
PPM1A
Protein phosphatase 1A
14q23.1






(formerly 2C), magnesium-






dependent, alpha isoform


227827_at
0.0008248
−4.5
ARGBP2
Arg/Abl-interacting protein
4q35.1






ArgBP2


227850_x_at
0.0001134
2.9
CDC42EP5
CDC42 effector protein (Rho
19q13.42






GTPase binding) 5






(CDC42EP5), mRNA.


227866_at
0.0007379
−2
RBM16
RNA binding motif protein 16
6q25.1-q25.3


227945_at
0.0003107
−2.3
TBC1D1
TBC1 (tre-2/USP6, BUB2,
4p14






cdc16) domain family, member






1 (TBC1D1), mRNA.


227952_at
0.0001513
4.1

Full length insert cDNA clone
4






YI46G04


227971_at
3.26E−05
−4.1
NRK
Nik related kinase (NRK),
Xq22.3






mRNA.


228012_at
0.0006848
−2
MATR3
Matrin 3
5q31.2


228027_at
3.77E−05
−4
GPRASP2
G protein-coupled receptor
Xq22.1






associated sorting protein 2






(GPRASP2), transcript variant






2, mRNA.


228030_at
0.0005637
3
RBM6
RNA binding motif protein 6
3p21.3


228098_s_at
0.0009902
2.2
MYLIP
myosin regulatory light chain
6p23-p22.3






interacting protein (MYLIP),






mRNA.


228202_at
0.0001345
−9.3
PLN
Phospholamban
6q22.1


228204_at
1.00E−07
2.9
PSMB4
proteasome (prosome,
1q21






macropain) subunit, beta type, 4






(PSMB4), mRNA.


228253_at
1.10E−05
3.8
LOXL3
lysyl oxidase-like 3 (LOXL3),
2p13






mRNA.


228297_at
0.0004293
3.3
CNN3
calponin 3, acidic (CNN3),
1p22-p21






mRNA.


228310_at
8.10E−06
−3.3
ENAH
enabled homolog (Drosophila)
1q42.12






(ENAH), transcript variant 2,






mRNA.


228331_at
0.0002123
3.4
C11orf31
Chromosome 11 open reading
11q12.1






frame 31


228333_at
0.0005407
−2.2

Full length insert cDNA clone
2






YT94E02


228335_at
0.0008785
−3.8
CLDN11
claudin 11 (oligodendrocyte
3q26.2-q26.3






transmembrane protein)






(CLDN11), mRNA.


228497_at
0.0001644
3.2
SLC22A15
solute carrier family 22 (organic
1p13.1






cation transporter), member 15






(SLC22A15), mRNA.


228523_at
0.0004047
2
NANOS1
nanos homolog 1 (Drosophila)
10q26.11






(NANOS1), transcript variant 2,






mRNA.


228551_at
3.95E−05
−3.1
MGC24039
Hypothetical protein
12p11.21






MGC24039


228554_at
1.35E−05
−4.7

MRNA; cDNA
11






DKFZp586G0321 (from clone






DKFZp586G0321)


228569_at
0.0006815
−2.2
PAPOLA
Poly(A) polymerase alpha
14q32.31


228573_at
0.0001917
−2.5
ANTXR2
Anthrax toxin receptor 2
4q21.21


228577_x_at
0.0003076
2.8
ODF2L
outer dense fiber of sperm tails
1p22.3






2-like (ODF2L), transcript






variant 2, mRNA.


228579_at
7.00E−07
5.2
KCNQ3
Potassium voltage-gated
8q24






channel, KQT-like subfamily,






member 3


228785_at
0.0006036
2.2
ZNF281
Zinc finger protein 281
1q32.1


228805_at
0.0003526
−3
FLJ44216
FLJ44216 protein (FLJ44216),
5q35.2






mRNA.


228841_at
0.0004789
−2

CDNA FLJ32429 fis, clone
5






SKMUS2001014


228850_s_at
0.0005294
2.8
SLIT2
Slit homolog 2 (Drosophila)
4p15.2


228885_at
1.82E−05
−4
MAMDC2
MAM domain containing 2
9q21.11






(MAMDC2), mRNA.


228905_at
0.0001087
−2.4

Transcribed locus, moderately
8






similar to XP_517655.1






PREDICTED: similar to






KIAA0825 protein [Pan






troglodytes]


228961_at
0.0004391
−2.1
FLJ35954
Hypothetical protein FLJ35954
5q11.2


229085_at
0.0003123
2.5
LRRC3B
leucine rich repeat containing
3p24






3B (LRRC3B), mRNA.


229114_at
0.0002683
−2
GAB1
GRB2-associated binding
4q31.21






protein 1


229119_s_at
0.0001491
−2.2
TTC19
Hypothetical protein
17p12






LOC125150


229129_at
0.0006968
−2.2
HNRPD
Heterogeneous nuclear
4q21.1-q21.2






ribonucleoprotein D (AU-rich






element RNA binding protein 1,






37 kDa)


229130_at
0.0002067
−3
LOC285535
Hypothetical protein
4p16.1






LOC285535


229145_at
4.45E−05
−4.2
C10orf104
chromosome 10 open reading
10q22.1






frame 104 (C10orf104), mRNA.


229160_at
0.0003552
−6.5
MUM1L1
melanoma associated antigen
Xq22.3






(mutated) 1-like 1 (MUM1L1),






mRNA.


229200_at
0.000156
2.3

Hypothetical LOC400813
1q44


229204_at
0.0003007
2.2
HP1-BP74
Heterochromatin protein 1,
1p36.12






binding protein 3


229218_at
0.0003816
3.3
COL1A2
Collagen, type I, alpha 2
7q22.1


229221_at
0.0006672
4.1
CD44
CD44 antigen (homing function
11p13






and Indian blood group system)


229287_at
0.0007653
−2.8

Full-length cDNA clone
14






CS0DK010YA20 of HeLa cells






Cot 25-normalized of Homo






sapiens (human)


229299_at
0.0001103
−2.2
FLJ30596
hypothetical protein FLJ30596
5p13.2






(FLJ30596), mRNA.


229308_at
2.94E−05
−5.4

Transcribed locus
18


229319_at
0.0001521
−2.4

Homo sapiens, clone
6






IMAGE: 4105966, mRNA


229331_at
0.0007515
2.3
SPATA18
spermatogenesis associated 18
4q11






homolog (rat) (SPATA18),






mRNA.


229339_at
0.0002425
−5
MYOCD
Myocardin
17p11.2


229354_at
0.000578
3.3
PDCD6
Aryl-hydrocarbon receptor
5pter-p15.2






repressor


229431_at
0.000216
−2.1
RFXAP
regulatory factor X-associated
13q14






protein (RFXAP), mRNA.


229483_at
0.0002012
2.5
UBE2H
Ubiquitin-conjugating enzyme
7q32






E2H (UBC8 homolog, yeast)


229515_at
0.0002226
−2.2
PAWR
PRKC, apoptosis, WT1
12q21






regulator


229520_s_at
0.0001174
2.3
C14orf118
Chromosome 14 open reading
14q22.1-q24.3






frame 118


229531_at
0.0007976
−2.2

Mitochondrial carrier triple
Xq22.2






repeat 6


229553_at
0.0008476
2
PGM2L1
phosphoglucomutase 2-like 1
11q13.4






(PGM2L1), mRNA.


229580_at
0.0008828
−6.5

Transcribed locus
3


229638_at
0.0008539
2.9
IRX3
iroquois homeobox protein 3
16q12.2






(IRX3), mRNA.


229642_at
0.0002914
4.2
ARHGEF7
Rho guanine nucleotide
13q34






exchange factor (GEF) 7






(ARHGEF7), transcript variant






2, mRNA.


229665_at
0.0001871
2.2
CSTF3
Hypothetical protein
11p13






LOC283267


229711_s_at
0.0003052
2.1

CDNA FLJ37519 fis, clone
12






BRCAN2004699


229748_x_at
0.0001962
2.7
LOC285458
Hypothetical protein
4






LOC285458


229795_at
0.0002666
3.4

Transcribed locus
12


229801_at
0.0001036
3.4
C10orf47
chromosome 10 open reading
10p14






frame 47 (C10orf47), mRNA.


229830_at
0.0004695
3.9
PDGFA
Platelet-derived growth factor
7p22






alpha polypeptide


229844_at
0.0001747
−2.2

Transcribed locus
3


229891_x_at
1.34E−05
−2.9
KIAA1704
KIAA1704
13q13-q14


229969_at
0.0003327
−2.5

Transcribed locus, moderately
6






similar to XP_508230.1






PREDICTED: zinc finger






protein 195 [Pan troglodytes]


229994_at
0.0002738
−3

MRNA; cDNA
1






DKFZp686J23256 (from clone






DKFZp686J23256)


230000_at
0.0004792
2.7
C17orf27
Chromosome 17 open reading
17q25.3






frame 27


230030_at
0.0003377
−5.2
HS6ST2
heparan sulfate 6-O-
Xq26.2






sulfotransferase 2 (HS6ST2),






mRNA.


230068_s_at
8.34E−05
−3.2
PEG3
paternally expressed 3 (PEG3),
19q13.4






mRNA.


230077_at
0.0001004
3.9
TFRC
Transferrin receptor (p90,
3q29






CD71)


230081_at
5.00E−07
−6.1
PLCXD3
phosphatidylinositol-specific
5p13.1






phospholipase C, X domain






containing 3 (PLCXD3),






mRNA.


230130_at
9.27E−05
3
SLIT2
Slit homolog 2 (Drosophila)
4p15.2


230141_at
0.0002042
−2
ARID4A
AT rich interactive domain 4A
14q23.1






(RBP1-like)


230174_at
0.0008233
−2.1
LYPLAL1
Lysophospholipase-like 1
1q41


230178_s_at
9.98E−05
−2.4
STATIP1
Signal transducer and activator
18q12.2






of transcription 3 interacting






protein 1


230270_at
7.57E−05
2.9

ESTs


230333_at
0.0008148
2.5
SAT
Spermidine/spermine N1-
Xp22.1






acetyltransferase


230336_at
0.0008645
2.7

Transcribed locus
4


230369_at
0.0005567
−2.1
GPR161
G protein-coupled receptor 161
1q24.2


230387_at
0.0001569
3.1
ATP2C1
ATPase, Ca++ transporting, type
3q22.1






2C, member 1


230440_at
2.81E−05
3.3
ZNF469
PREDICTED: zinc finger
16






protein 469 (ZNF469), mRNA.


230561_s_at
0.0001454
−2.9
FLJ23861
hypothetical protein FLJ23861
2q34






(FLJ23861), mRNA.


230574_at
0.0008056
2.9

Hypothetical LOC388480
18q21.33


230746_s_at
1.50E−06
13.3
STC1
stanniocalcin 1 (STC1), mRNA.
8p21-p11.2


230758_at
0.00064
−2.6

Transcribed locus
X


230793_at
7.34E−05
−3.1
LRRC16
leucine rich repeat containing 16
6p22.2






(LRRC16), mRNA.


230850_at
0.0001807
3
FMNL3
Formin-like 3
12q13.12


230885_at
0.0001271
2.2
SPG7
Spastic paraplegia 7, paraplegin
16q24.3






(pure and complicated






autosomal recessive)


230958_s_at
4.15E−05
−2.1

MRNA; cDNA
1






DKFZp686J23256 (from clone






DKFZp686J23256)


231130_at
0.0009273
−2
FKBP7
FK506 binding protein 7
2q31.2


231183_s_at
0.000129
4.3
JAG1
jagged 1 (Alagille syndrome)
20p12.1-p11.23






(JAG1), mRNA.


231202_at
0.0002962
3.1
FLJ38508
Aldehyde dehydrogenase 1
12q23.3






family, member L2


231411_at
0.0001224
3.2
LHFP
lipoma HMGIC fusion partner
13q12






(LHFP), mRNA.


231597_x_at
0.0001876
15.9

ESTs, Weakly similar to T47135






hypothetical protein






DKFZp761L0812.1 [H. sapiens]


231806_s_at
0.0009603
2.1
STK36
serine/threonine kinase 36
2q35






(fused homolog, Drosophila)






(STK36), mRNA.


231825_x_at
8.13E−05
3.6
ATF7IP
Activating transcription factor 7
12p13.1






interacting protein


231882_at
0.0002463
2.7

CDNA FLJ10674 fis, clone
22






NT2RP2006436


231886_at
0.0003613
3

Similar to hypothetical protein
3q29






LOC284701


232034_at
0.0008165
3.9
LOC203274
Hypothetical protein
9q21.11






LOC203274


232145_at
0.0002539
2.1
LOC388969
hypothetical LOC388969
2p11.2






(LOC388969), mRNA.


232150_at
2.29E−05
3.1
C20orf18
Chromosome 20 open reading
20p13






frame 18


232169_x_at
0.0002893
3.1
NDUFS8
NADH dehydrogenase
11q13






(ubiquinone) Fe—S protein 8,






23 kDa (NADH-coenzyme Q






reductase)


232174_at
7.00E−06
4.1
EXT1
Exostoses (multiple) 1
8q24.11-q24.13


232180_at
8.56E−05
3.2
UGP2
UDP-glucose
2p14-p13






pyrophosphorylase 2


232215_x_at
0.0001855
3.4
FLJ11029
Hypothetical protein FLJ11029
17q23.2


232254_at
8.75E−05
2.5
FBXO25
F-box protein 25
8p23.3


232266_x_at
0.0001426
3.9
CDC2L5
Cell division cycle 2-like 5
7p13






(cholinesterase-related cell






division controller)


232304_at
2.60E−06
4
PELI1
Pellino homolog 1 (Drosophila)
2p13.3


232347_x_at
0.0001636
2.6
CBR4
Carbonic reductase 4
4q32.3


232406_at
0.0001583
2.9
JAG1
Jagged 1 (Alagille syndrome)
20p12.1-p11.23


232458_at
1.67E−05
4.1
COL3A1
Collagen, type III, alpha 1
2q31






(Ehlers-Danlos syndrome type






IV, autosomal dominant)


232516_x_at
7.53E−05
3.3
YAP
YY1 associated protein 1
1q22


232530_at
0.0006114
2.8
PLD1
Phospholipase D1,
3q26






phophatidylcholine-specific


232538_at
0.0008933
2.4

CDNA: FLJ23573 fis, clone
16






LNG12520


232541_at
0.0007593
3.5
EGFR
Epidermal growth factor
7p12






receptor (erythroblastic






leukemia viral (v-erb-b)






oncogene homolog, avian)


232617_at
8.18E−05
3.2
CTSS
cathepsin S (CTSS), mRNA.
1q21


232653_at
0.000473
3.5

Homo sapiens cDNA FLJ14044






fis, clone HEMBA1006124


232702_at
0.0006941
3
RABGAPIL
RAB GTPase activating protein
1q24






1-like


232797_at
0.0008213
2.3
ITGAV
Integrin, alpha V (vitronectin
2q31-q32






receptor, alpha polypeptide,






antigen CD51)


232814_x_at
0.0005021
2.8
C14orf153
Chromosome 14 open reading
14q32.32-q32.33






frame 153


232889_at
0.0003899
2.6

CDNA clone IMAGE: 5576908
5


232952_at
0.0001405
2.7
DDEF1
HSPC054 protein
8q24.1-q24.2


233041_x_at
0.0004112
3
BTBD9
BTB (POZ) domain containing 9
6p21


233180_at
1.17E−05
2.8
RNF152
Ring finger protein 152
18q21.33


233274_at
3.00E−07
3.6
NCK1
NCK adaptor protein 1
3q21


233319_x_at
0.0001543
3.1
PHACTR4
Phosphatase and actin regulator 4
1p35.3


233330_s_at
4.59E−05
5.6

Similar to Ribosome biogenesis
9q13






protein BMS1 homolog


233406_at
0.0002846
2.9
KIAA0256
KIAA0256 gene product
15q21.1


233496_s_at
0.0003586
−2
CFL2
Cofilin 2 (muscle)
14q12


233702_x_at
0.0007629
3.1

CDNA: FLJ20946 fis, clone
7






ADSE01819


233849_s_at
0.0005935
−2.3
ARHGAP5
Rho GTPase activating protein 5
14q12






(ARHGAP5), transcript variant






2, mRNA.


233912_x_at
1.73E−05
4.4
ELMOD2
ELMO domain containing 2
4q31.21


234192_s_at
0.0001567
−3.1
GKAP1
G kinase anchoring protein 1
9q21.32






(GKAP1), mRNA.


234339_s_at
6.34E−05
−2.4
GLTSCR2
glioma tumor suppressor
19q13.3






candidate region gene 2






(GLTSCR2), mRNA.


234464_s_at
4.21E−05
3
EME1
essential meiotic endonuclease 1
17q21.33






homolog 1 (S. pombe) (EME1),






mRNA.


234512_x_at
2.40E−06
−3.1
LOC442159
PREDICTED: similar to Rpl7a
6






protein (LOC442159), mRNA.


234562_x_at
1.80E−06
8.6
CKLFSF8
Chemokine-like factor super
3p23






family 8


234578_at
7.26E−05
4.9

MRNA; cDNA
1






DKFZp434E1812 (from clone






DKFZp434E1812)


234675_x_at
6.52E−05
4.3

CDNA: FLJ23566 fis, clone
14






LNG10880


234723_x_at
1.50E−06
9.4

CDNA: FLJ21228 fis, clone
7






COL00739


234753_x_at
0.0002577
5.7


234762_x_at
8.75E−05
4.1
NLN
Neurolysin (metallopeptidase
5q12.3






M3 family)


234788_x_at
0.0001541
3.3
FLJ13611
Hypothetical protein FLJ13611
5q12.3


234873_x_at
0.000607
−2


234981_x_at
9.85E−05
4.4
LOC134147
Similar to mouse
5p15.2






2310016A09Rik gene


234985_at
9.30E−06
2.9
LOC143458
Hypothetical protein
11p13






LOC143458


234998_at
0.0009467
−2.7

CDNA clone IMAGE: 5313062
15


235005_at
0.0003344
−2.3
MGC4562
Hypothetical protein MGC4562
15q22.31


235061_at
0.0002578
−2.9
PPM1K
protein phosphatase 1K (PP2C
4q22.1






domain containing) (PPM1K),






mRNA.


235072_s_at
6.66E−05
−3

Transcribed locus
6


235122_at
5.42E−05
3.1

CDNA clone IMAGE: 6254031
1


235151_at
0.0004926
−2.1
LOC283357
Hypothetical protein
12p13.33






LOC283357


235204_at
1.87E−05
3.1
ENTPD7
Ectonucleoside triphosphate
10






diphosphohydrolase 7


235205_at
0.0001252
3.5
LOC346887
PREDICTED: similar to solute
8






carrier family 16






(monocarboxylic acid






transporters), member 14






(LOC346887), mRNA.


235278_at
0.0002619
−3.6
C20orf133
chromosome 20 open reading
20p12.1






frame 133 (C20orf133),






transcript variant 2, mRNA.


235309_at
p < 1e−07
−4.9

CDNA clone IMAGE: 4140029
16


235327_x_at
0.0002751
2.9
UBXD4
UBX domain containing 4
2p23.3






(UBXD4), mRNA.


235343_at
3.96E−05
4.2
FLJ12505
Hypothetical protein FLJ12505
1q32.3


235374_at
3.04E−05
2.7
MDH1
Malate dehydrogenase 1, NAD
2p13.3






(soluble)


235412_at
0.0002124
3.2
ARHGEF7
Rho guanine nucleotide
13q34






exchange factor (GEF) 7


235433_at
0.0003304
−2.2
SATL1
Spermidine/spermine N1-acetyl
Xq21.1






transferase-like 1


235556_at
0.0001833
−2

Transcribed locus, weakly
5






similar to NP_703324.1






glutamic acid-rich protein (garp)






[Plasmodium falciparum 3D7]


235601_at
0.0003888
2.7

ESTs


235612_at
2.90E−05
−3.1

Transcribed locus, moderately
1






similar to NP_858931.1 NES1






nitrogen fixation 1 isoform b






precursor; cysteine desulfurase;






nitrogen-fixing bacteria S-like






protein; nitrogen fixation 1 (S. cerevisiae,






homolog) [Homo






sapiens]


235628_x_at
0.0004866
2.4

Hypothetical protein
5q23.2






LOC133926


235693_at
0.0003764
2.9

Transcribed locus
5


235725_at
0.0004132
−2.2

Transcribed locus
14


235927_at
6.41E−05
2.4
XPO1
Exportin 1 (CRM1 homolog,
2p16






yeast)


235944_at
0.0004031
3.3
HMCN1
hemicentin 1 (HMCN1),
1q25.3-q31.1






mRNA.


236249_at
1.54E−05
2.7
IKIP
IKK interacting protein (IKIP),
12q23.1






transcript variant 1, mRNA.


236251_at
8.16E−05
2.7
ITGAV
Integrin, alpha V (vitronectin
2q31-q32






receptor, alpha polypeptide,






antigen CD51)


236678_at
0.0005987
2.8
JAG1
Jagged 1 (Alagille syndrome)
20p12.1-p11.23


236715_x_at
0.0002206
2.5
UACA
uveal autoantigen with coiled-
15q22-q24






coil domains and ankyrin repeats






(UACA), transcript variant 1,






mRNA.


236829_at
6.57E−05
2.9
TMF1
TATA element modulatory
3p21-p12






factor 1


236883_at
0.0003076
2.6

ESTs


236936_at
0.0007927
−2.4

Transcribed locus
8


236966_at
0.0004757
2.6
TXNDC6
thioredoxin domain containing 6
3q22.3






(TXNDC6), mRNA.


236974_at
0.0003599
2.5
CCNI
Cyclin I
4q21.1


237206_at
0.0003483
−5.9
MYOCD
Myocardin
17p11.2


237333_at
0.0008796
−2
SYNCOILIN
Syncoilin, intermediate filament 1
1p34.3-p33


237475_x_at
0.0001562
4.3
SEPP1
Selenoprotein P, plasma, 1
5q31


237494_at
2.03E−05
3.2

Transcribed locus
15


237868_x_at
0.000358
3

ESTs, Weakly similar to






ALUF_HUMAN !!!! ALU






CLASS F WARNING ENTRY






!!! [H. sapiens]


238026_at
0.0007905
−2
RPL35A
Ribosomal protein L35a
3q29-qter


238142_at
0.000678
2.4
LOC253982
Hypothetical protein
16q11.2






LOC253982


238183_at
0.0001644
3.7

ESTs


238273_at
0.0007281
2

Full-length cDNA clone
7






CS0DB005YG10 of






Neuroblastoma Cot 10-






normalized of Homo sapiens






(human)


238327_at
6.09E−05
2.6

Similar to MGC52679 protein
22q13.33


238478_at
0.000183
−3.7
BNC2
Basonuclin 2
9p22.3-p22.2


238584_at
0.0002196
3.3
IQCA
IQ motif containing with AAA
2q37.2-q37.3






domain


238613_at
8.97E−05
−2.7
ZAK
sterile alpha motif and leucine
2q24.2






zipper containing kinase AZK






(ZAK), transcript variant 2,






mRNA.


238642_at
0.0003128
3
LOC338692
Ankyrin repeat domain 13
11q13.2






family, member D


238673_at
6.02E−05
4.7

Transcribed locus
8


238714_at
0.0001825
2.5

RAB12, member RAS oncogene
18p11.22






family


238719_at
0.0004981
−2.5
PPP2CA
Protein phosphatase 2 (formerly
5q31.1






2A), catalytic subunit, alpha






isoform


238852_at
0.0003011
2.3

ESTs


238878_at
0.0006945
−6.2
ARX
Aristaless related homeobox
Xp22.1-p21.3


239227_at
9.04E−05
3
EXT1
Exostoses (multiple) 1
8q24.11-q24.13


239246_at
0.0002103
3
FARP1
FERM, RhoGEF (ARHGEF)
13q32.2






and pleckstrin domain protein 1






(chondrocyte-derived)


239258_at
0.0009901
3.1
RHOQ
Ras homolog gene family,
2p21






member Q


239262_at
3.72E−05
−3.8

CDNA FLJ26242 fis, clone
11






DMC00770


239264_at
0.0003642
2.2
SEC8L1
SEC8-like 1 (S. cerevisiae)
7q31


239367_at
0.0001649
3.1
BDNF
brain-derived neurotrophic
11p13






factor (BDNF), transcript variant






6, mRNA.


239516_at
0.0001834
2.7
LYPLAL1
Lysophospholipase-like 1
1q41


239540_at
0.0009106
2.6
GTF3C1
General transcription factor IIIC,
16p12






polypeptide 1, alpha 220 kDa


239748_x_at
0.0003286
3.7
OCIA
ovarian carcinoma
4p11






immunoreactive antigen


239806_at
0.000252
4.4

Transcribed locus
2


239848_at
0.0005816
−2.7
GA17
Dendritic cell protein
11p13


240216_at
0.0001639
2.6
ZBTB20
Zinc finger and BTB domain
3q13.2






containing 20


240421_x_at
0.0001971
3.7

CDNA clone IMAGE: 5268630
4


240655_at
3.01E−05
4.3
ALCAM
Activated leukocyte cell
3q13.1






adhesion molecule


240795_at
0.0001514
2.3

CDNA clone IMAGE: 5288566
5


241223_x_at
0.0002281
3.2

ESTs, Weakly similar to






ALU1_HUMAN ALU






SUBFAMILY J SEQUENCE






CONTAMINATION






WARNING ENTRY






[H. sapiens]


241268_x_at
0.0008446
3.5
SAMHD1
SAM domain and HD domain 1
20pter-q12


241303_x_at
0.0007123
2.9

ESTs


241387_at
0.0008125
2.7
PTK2
PTK2 protein tyrosine kinase 2
8q24-qter


241421_at
0.0005012
2.6

Transcribed locus
1


241435_at
1.52E−05
4.4
ETS1
V-ets erythroblastosis virus E26
11q23.3






oncogene homolog 1 (avian)


241617_x_at
0.000334
2.6

ESTs, Weakly similar to






810024C cytochrome oxidase I






[H. sapiens]


241627_x_at
0.0005548
2.9
FLJ10357
Hypothetical protein FLJ10357
14q11.2


241632_x_at
3.70E−06
3.3

ESTs


241686_x_at
3.28E−05
4.4

ESTs, Weakly similar to






hypothetical protein FLJ20378






[Homo sapiens] [H. sapiens]


241718_x_at
7.37E−05
3.4

ESTs


241727_x_at
0.0003166
2.3
DHFRL1
dihydrofolate reductase-like 1
3q11.2






(DHFRL1), mRNA.


241769_at
0.0004802
2.5
ITGAV
Integrin, alpha V (vitronectin
2q31-q32






receptor, alpha polypeptide,






antigen CD51)


241809_at
0.0001618
2.5
LOC284465
Hypothetical protein
1p13.2






LOC284465


242029_at
9.60E−06
3.7
FNDC3B
Fibronectin type III domain
3q26.31






containing 3B


242051_at
6.20E−06
−3.7

Transcribed locus
X


242100_at
2.40E−06
4.8
CSS3
chondroitin sulfate synthase 3
5q23.3






(CSS3), mRNA.


242171_at
0.0007535
3.1

ESTs, Weakly similar to






ALU1_HUMAN ALU






SUBFAMILY J SEQUENCE






CONTAMINATION






WARNING ENTRY






[H. sapiens]


242233_at
7.63E−05
2.4
KIAA1219
KIAA1219 protein
20q11.23


242240_at
0.0001413
3
PTK2
PTK2 protein tyrosine kinase 2
8q24-qter


242329_at
8.94E−05
3.1
LOC401317
PREDICTED: hypothetical
7






LOC401317 (LOC401317),






mRNA.


242363_at
9.05E−05
−2.7
DNCI2
Dynein, cytoplasmic,
2q31.1






intermediate polypeptide 2


242364_x_at
0.0001237
3.1
EVER1
Epidermodysplasia
17q25.3






verruciformis 1


242369_x_at
7.20E−06
4.1
NCOA2
Nuclear receptor coactivator 2
8q13.3


242398_x_at
3.00E−06
6.1
MEP50
WD repeat domain 77
1p13.2


242405_at
4.08E−05
2
MAML2
Mastermind-like 2 (Drosophila)
11q21


242488_at
2.11E−05
−4.7

CDNA FLJ38396 fis, clone
1






FEBRA2007957


242500_at
0.0007278
2.6

Transcribed locus
6


242546_at
2.70E−06
5.4

LOC440156
14q11.1


242578_x_at
0.0002453
4
SLC22A3
Solute carrier family 22
6q26-q27






(extraneuronal monoamine






transporter), member 3


242862_x_at
6.29E−05
5.2

ESTs


242999_at
0.0007732
2.3
ARHGEF7
Rho guanine nucleotide
13q34






exchange factor (GEF) 7






(ARHGEF7), transcript variant






2, mRNA.


243_g_at
9.74E−05
2.1
MAP4
microtubule-associated protein 4
3p21






(MAP4), transcript variant 2,






mRNA.


243006_at
1.10E−06
4.7
FYN
FYN oncogene related to SRC,
6q21






FGR, YES


243147_x_at
7.12E−05
5.7

ESTs, Weakly similar to






RMS1_HUMAN REGULATOR






OF MITOTIC SPINDLE






ASSEMBLY 1 [H. sapiens]


243249_at
7.21E−05
2.4

ESTs, Weakly similar to






hypothetical protein FLJ20378






[Homo sapiens] [H. sapiens]


243305_at
0.0003329
2.9
KIAA1340
Kelch domain containing 5
12p11.22


243442_x_at
0.0001001
3.7

ESTs, Weakly similar to






ALU1_HUMAN ALU






SUBFAMILY J SEQUENCE






CONTAMINATION






WARNING ENTRY






[H. sapiens]


243846_x_at
0.0007887
3
FLJ32810
Hypothetical protein FLJ32810
11q22.1


243915_at
0.0001482
2.9

ESTs, Weakly similar to






2109260A B cell growth factor






[H. sapiens]


244007_at
0.000373
−2

Transcribed locus
9


244022_at
0.0007683
2
FNDC3B
Fibronectin type III domain
3q26.31






containing 3B


244050_at
0.0001908
−2.6
LOC401494
similar to RIKEN 4933428I03
9p21.3






(LOC401494), mRNA.


244188_at
0.0004372
3.1
FLJ21924
Hypothetical protein FLJ21924
11p13


244193_at
0.0003257
2.1
FLJ13236
Hypothetical protein FLJ13236
12q13.12


244197_x_at
2.30E−05
3.3
CNOT2
CCR4-NOT transcription
12q15






complex, subunit 2


244457_at
8.66E−05
3.1
ITPR2
Inositol 1,4,5-triphosphate
12p11






receptor, type 2


244503_at
0.0004105
2.5

ESTs


244633_at
0.000111
2.5
PIAS2
Protein inhibitor of activated
18q21.1






STAT, 2


244648_at
0.0003403
3.5
FLJ10996
Hypothetical protein FLJ10996
2q14.1


244745_at
0.0006739
−2.7
RERG
RAS-like, estrogen-regulated,
12p12.3






growth inhibitor (RERG),






mRNA.


31874_at
8.21E−05
2.9
GAS2L1
Growth arrest-specific 2 like 1
22q12.2


33323_r_at
0.0001426
5.1
SFN
Stratifin
1p36.11


38069_at
0.0003197
2
CLCN7
chloride channel 7 (CLCN7),
16p13






mRNA.


38671_at
0.0003864
2.4
PLXND1
plexin D1 (PLXND1), mRNA.
3q21.3


39549_at
0.0008832
2.5
NPAS2
neuronal PAS domain protein 2
2q11.2






(NPAS2), mRNA.


39891_at
0.0005541
2.2
DKFZp547K1113
Hypothetical protein
15q26.1






DKFZp547K1113


40524_at
3.70E−05
2.8
PTPN21
protein tyrosine phosphatase,
14q31.3






non-receptor type 21 (PTPN21),






mRNA.


41856_at
0.0008627
2.3
UNC5B
Unc-5 homolog B (C. elegans)
10q22.1


44783_s_at
6.85E−05
3
HEY1
hairy/enhancer-of-split related
8q21






with YRPW motif 1 (HEY1),






mRNA.


46665_at
5.11E−05
2.4
SEMA4C
sema domain, immunoglobulin
2q11.2






domain (Ig), transmembrane






domain (TM) and short






cytoplasmic domain,






(semaphorin) 4C (SEMA4C),






mRNA.


47550_at
3.40E−05
4.4
LZTS1
leucine zipper, putative tumor
8p22






suppressor 1 (LZTS1), mRNA.


50376_at
0.000154
2.1
ZNF444
zinc finger protein 444
19q13.43






(ZNF444), mRNA.


52255_s_at
1.15E−05
6.1
COL5A3
collagen, type V, alpha 3
19p13.2






(COL5A3), mRNA.


55583_at
0.0001374
3.2
DOCK6
dedicator of cytokinesis 6
19p13.2






(DOCK6), mRNA.


57539_at
0.0006736
2.1
ZGPAT
zinc finger, CCCH-type with G
20q13.3






patch domain (ZGPAT),






transcript variant 3, mRNA.


57703_at
0.0001957
2.4
SENP5
SUMO1/sentrin specific
3q29






peptidase 5 (SENP5), mRNA.


57739_at
0.0004289
2.2
DND1
Dead end homolog 1 (zebrafish)
5q31.3


59433_at
5.44E−05
2.2

Transcribed locus
X


61734_at
3.36E−05
2.7
RCN3
Reticulocalbin 3, EF-hand
19q13.33






calcium binding domain









As indicated in Table 1, 652 genes were up-regulated ≧2-fold in ovarian cancer endothelium samples. Of the 652 genes that were up-regulated ≧2-fold, 35 genes were elevated at least 6-fold in tumor endothelium (Table 2), with 7 being elevated more than 10-fold and 2 being elevated more than 28-fold.







TABLE 2







Genes up-regulated by ≧6-fold in the tumor associated endothelium.












Entrez


Fold
chromosomal



Gene ID
Gene
Description
difference
location
Function















25975
EGFL6
EGF-like-domain,
36.8
Xp22
May regulate cell cycle and




Multiple 6


oncogenesis


7130
TNFAIP6
Tumor necrosis factor,
29.1
2q23.3
Anti-inflammatory and




Alpha-induced protein 6


chondroprotective effect


7291
TWIST1
Basic helix-loop-helix
19.0
7p21.2
Inhibits chondrogenesis




(bHLH) transcription




factor


6781
STC1
Stanniocalcin 1
13.3
8p21-p11.2
Regulates calcium/







phosphate homeostasis, and







cell metabolism


84525
HOP
Homeodomain-only
13.1
4q11-q12
Transcriptional repressor.




Protein, transcript


Modulates serum response




variant 2


factor-dependent cardiac-







specific gene expression and







cardiac development


1462
CSPG2
Chondroitin sulfate
10.4
5q14.3
Extracellular matrix




proteoglycan 2


component of the vitreous gel.




(versican)


Anti-cell adhesive.


57125
PLXDC1
Plexin domain
10.2
17q21.1
Unknown




containing 1


6696
SPP1
Secreted
9.5
4q21-q25
Expressed during




phosphoprotein 1


embryogenesis, wound




(osteopontin, bone


healing, and tumorigenesis.




sialoproteinI,


Regulates the assembly of




Early T-lymphocyte


heterotypic fibers composed of




activation1)


both type 1 and type V







collagen.


4318
MMP9
Matrix metallopeptidase 9
9.4
20q11.2-q13.1
Breakdown of extracellular




(gelatinase B, 92 kDa


matrix. Plays a role in




type IV collagenase)


angiogenesis and tumor







invasion


3937
LCP2/SLP76
Lymphocyte cytosolic
8.7
5q33.1-qter
Promotes T-cell




protein 2 (SH2 domain


development and




containing Leukocyte


activation




protein of 76 kDa)


152189
CKLFSF8
Chemokine-like factor
8.6
3p23
Regulates EGF-induced




Superfamily 8


signaling. Regulates cell







proliferation


5366
PMAIP1
Phorbol-12-myristate-
8.5
18q21.32
Unknown




13-acetate-induced




protein 1


24147
FJX1
Four jointed box 1
7.7
11p13
In drosophila, a downstream




(drosophila)


target of the Notch signaling







pathway, regulates cell growth







and differentiation. Not known







in human


8038
ADAM12
ADAM metallo-
7.6
10q26.3
Critical for tumor




peptidase domain 12


development. Involved in cell-




(meltrin alpha)


cell and cell-matrix







interactions.


9636
GIP2
Interferon, alpha-
6.9
1p36.33
Unclear, may be related to




inducible protein (clone


regulation of cell proliferation




IFI-15K)


and differentiation


25878
MXRA5
Matrix-remodeling
6.9
Xp22.33
Matrix remodeling




associated 5


1123
CNH1
Chimerin
6.9
2q31-q32.1
Rho GTPase activating protein




(chimaerin) 1


3310
HSPA6
Heat shock 70 KDa
6.8
1q23
Involved in protein




protein 6 (HSP70B)


conformational interactions


11211
FZD10
Frizzled homolog 10
6.7
12q24.33
Receptor for the wingless




(Drosophila)


Type MMTV integration







site family.


10631
POSTN
Periostin, osteoblast
6.7
13q13.3
Promotes integrin-dependent




specific factor


cell adhesion and motility,







involved in extracellular







matrix deposition


85477
SCIN
Scinderin
6.6
7p21.3
Ca2+ dependent actin filament







severing protein, regulates







cortical actin network







dynamics


27242
TNFRSF21
Tumor necrosis factor
6.6
6p21.1-12.2
Unclear; maybe related to




Receptor superfamily,


activation of NF-kappaB and




Member 21


MAPK8/JNK, induces cell







apoptosis, involved in







inflammation and immune







regulation.


25891
DKFZP586-
Regeneration associated
6.2
11p13
Unknown



H2123
muscle protease,




transcript variant 2


4582
MUC1
Mucin 1,
6.2
1q21
Regulates cell aggregation,




transmembrane,


adhesion




transcript variant 4


79084
MEP50
WD repeat domain 77,
6.1
1p13.2
Involved in the methylation




WDR77


and assembly of spliceosomal







snRNAs Sm proteins


50509
COL5A3
Collagen, type V, alpha 3
6.1
19p13.2
Extracellular protein,







associated with formation of







fibrils, and some connective







tissue pathology such as







inflammation, cancer and







atherosclerosis


6205
RPS11
Ribosomal protein S11
6.1
19q13.3
Involved in the recognition of







termination codons.


55803
CENTA2
Centaurin, alpha 2
6.1
17q11.2
A phosphatidylinositide-







binding protein present in the







dense membrane fractions of







cell extracts


2295
FOXF2
Forkhead box F2
6.0
6p25.3
Regulates cell proliferation







and survival, associated with







BMP and Wnt signaling
















TABLE 3







Genes down-regulated in tumor associated endothelium.












Entrez



Chromosomal



Gene ID
Gene
Description
Fold difference
location
Function















5350
PLN
Phospholamban
0.108
6q22.1
Inhibits sarcoplasmic







reticulum Ca(2+)-







ATPase activity


6401
SELE
Selectin E,
0.112
1q22-q25
Cell surface lycoprotein.




endothelial


Inhibits cell adhesion.




Adhesion molecule 1


Early marker of







inflammation


9687
GREB1
GREB1 protein,
0.116
2p25.1
Transcription factor,




transcript variant a


inhibits cell proliferation


4969
OGN
Osteoglycin
0.147
9q22
Regulates collagen




osteoinductive


fibrillogenesis




Factor, mimecan),




transcript




variant 3


25890
AB13BP
ABI gene family,
0.152
3q12
May play a role in cell




member 3 (NESH)


motility by regulating




binding protein


NESH function


90161
HS6ST2
Heparn sulfate 6-
0.153
Xq26.2
Plays a role in growth




O-sulfotransferase 2


factor signaling, cell







adhesion, and







enzymatic catalysis.







Maybe involved in







vascularization by







mediating FGF signaling


139221
MUM1L1
Melanoma associated
0.155
Xq22.3
Encodes tumor specific




antigen (mutated) 1-


antigens




like 1


4129
MAOB
Monoamine oxidase B
0.156
Xp11.23
Regulates







neurotransmitters in







central nervous system


9452
ITM2A
Integral membrane
0.156
Xq13.3-Xq21.2
Transmembrane protein.




protein 2A


Marker of early stage of







endochondral







ossification


170302
ARX
Aristaless related
0.162
Xp22.1-p21.3
Organ development.




homeobox


Bifunctional







transcription factor


10659
CUGBP2
CUG triplet repeat,
0.163
10p13
Binds and stabilizes




RNA binding Protein


COX2 mRNA, inhibits




2, transcript variant 2


its translation


5577
PRKAR2B
Protein kinase,
0.163
7q22
Encodes a regulatory




cAMP-dependent


subunit RII beta of




regulatory, type II,


human cAMP-dependent




beta


protein







kinase A


345557
LCXD3
Phosphatidylinositol-
0.165
5p13.1
Quantitatively




specific


solubilizes




phospholipase C, X


AChE from purified




domain containing 3


synaptic plasma







membranes and







intact synaptosomes of







Torpedo ocellata electric







organ









Multiple genes encoding proteins involved in extracellular matrix function, such as collagens, TNFAIP6, ADAMTS4, MMP9, MMP11, had increased expression in tumor vasculature compared with normal ovarian vasculature. The αv integrin (vitronectin receptor) was elevated 2.5-fold in tumor endothelium. Several transcription factors were upregulated in the ovarian cancer vasculature. For example, HEYL was increased 3-fold. In addition, several transcription factors were identified including E2F transcription factor 3 (E2F3; plays a role in cell proliferation) (Black, Proc Natl Acad Sci U.S.A. 102: 15948-15953, 2005); runt-related transcription factor 1 (RUNX1; plays a role in angiogenesis) (Iwatsuki et al., Oncogene 24: 1129-1137, 2005), signal transducer and activator of transcription 2 (STAT2; role in cellular proliferation) (Gomez and Reich, J. Immunol. 170: 5373-5381, 2003), the SNAIL-related zinc-finger transcription factor, SLUG (SNAI2) (Perez-Mancera et al., Oncogene 24: 3073-3082, 2005), and Twist1 (Mironchik et al. Cancer Res. 65: 10801-10809, 2005). These genes were elevated 2-18 fold in the ovarian cancer vasculature relative to normal ovarian endothelial cells.
Additional genes were identified as being overexpressed in ovarian tumor endothelial cells that had previously been reported to be overexpressed in tumor cells. For example, epidermal growth factor receptor (EGFR) expression was increased by 3.5-fold in the tumor endothelium. EGFR is known to be overexpressed in ovarian carcinomas and is predictive of poor outcome (Berchuck et al., Am. J. Obstet. Gynecol. 164: 669-674, 1991). Similarly, non-receptor kinases such as focal adhesion kinase (FAK or PTK2; 3.1-fold increase) and Fyn (4.7 fold increase), which are play functional roles for tumor cells were detected. Genes that are overexpressed on both tumor cells and tumor-associated endothelial cells are targets for anti-vascular therapy due to the ability to target both the epithelial and stromal compartments.
In addition to the 652 genes that were identified as being up-regulated in ovarian tumor endothelial cells, 497 genes were down-regulated ≧2-fold in ovarian cancer endothelium samples (Table 1). FIG. 1 illustrates the fold changes observed in the relative expression levels between microarray data and real-time quantitative RT-PCR data from the pro-angiogenic gene signature provided in Table 1. Of the 497 genes that were down-regulated ≧2-fold, 17 genes were decreased at least 6-fold (as provide in Table 3). For example, monoamine oxidase B (MAOB), a gene responsible for detoxification and degradation of monoamines was decreased by 6.4-fold in the tumor endothelial cells (Grimsby et al., Nat. Genet. 17: 206-210, 1997). Decorin, a small multi-functional proteoglycan with anti-angiogenic properties, was decreased by 4.8-fold (Sulochana et al., J. Biol. Chem. 280: 27935-27948, 2005). Several other genes with potential anti-angiogenic or anti-proliferative roles such as Fibulin-5 (FBLN-5) and checkpoint suppressor 1 (CHES1) were down-regulated by 4.5-fold and 4.3-fold, respectively (Albig and Schiemann, DNA Cell Biol. 23: 367-379, 2004; and Scott and Plon, Gene 359: 119-126, 2005). These findings indicate that tumor endothelial cell and non-tumor endothelial cell isolates possess distinct expression profiles.
Example 4
Identification of Tumor Endothelial Markers
This example provides specific tumor endothelial cell markers.
Differentially regulated genes expressed in tumor-associated endothelium were identified by comparing tumor-associated endothelium versus normal endothelium with tumor-associated epithelial cells versus ovarian surface epithelium (OSE). The expression profile of microdissected papillary serous ovarian cancers using the same microarray methods has previously been reported (Bonome et al. Cancer Res. 65: 10602-10612, 2005). The current disclosed list of differentially expressed genes in tumor-associated endothelial cells was compared with the gene list identified for laser microdissected tumor-epithelial cells. A total of 534 differentially regulated genes were uniquely altered (up- or down-regulated) in the endothelial cells. The 28 genes with the greatest level of increase in ovarian tumor endothelial cells are listed in Table 4, of which 23 genes had a ≧6-fold increase in expression. A complete listing of the 534 differentially regulated genes is provided in Table 5. Further, FIG. 2 illustrates protein expression levels detected in ovarian endothelial cells following staining of samples with immunofluorescently-labeled PTK2, Fyn, MMP-9, β2-arrestin, Jagged1 and PLXDC1, respectively.
These findings identify tumor endothelial cell specific genes that can be used as biomarkers and potential targets for treatment of ovarian cancer.







TABLE 4







Genes specifically regulated in tumor-endothelium












Entrez


Fold
Chromosomal



Gene ID
Gene
Description
difference
location
Function















25975
EGFL6
EGF-like-domain, multiple 6
36.848
Xp22
May regulate cell cycle




(EGFL6)


and oncogenesis


7130
TNFAIP6
Tumor necrosis factor, alpha-
29.062
2q23.3
Anti-inflammatory and




induced protein 6 (TNFAIP6)


chondroprotective effect


7291
TWIST1
Twist homolog 1
18.969
7p21.2
Inhibits chondrogenesis




(acrocephalosyndactyly 3;




Saethre-Chotzen syndrome)




(Drosophila) (TWIST1)


6781
STC1
Stanniocalcin 1 (STC1)
13.326
8p21-p11.2
Regulates calcium/







phosphate homeostasis,







and cell metabolism


84525
HOP
Homeodomain-only protein
13.144
4q11-q12
Transcriptional




(HOP), transcript variant 2


repressor. Modulates







serum response







factor-dependent







cardiac-specific gene







expression and cardiac







development


1462
CSPG2
Chondroitin sulfate
10.355
5q14.3
Extracellular matrix




proteoglycan 2 (versican)


component of the




(CSPG2)


vitreous gel. Anti-cell







adhesive.


57125
PLXDC1
Plexin domain containing 1
10.215
17q21.1
Unknown




(PLXDC1)


4318
MMP9
Matrix metallopeptidase 9
9.389
20q11.2-q13.1
Breakdown of




(gelatinase B, 92 kDa


extracellular




gelatinase, 92 kDa type IV


matrix. Plays a role in




collagenase) (MMP9)


angiogenesis and tumor







invasion


3937
LCP2
Lymphocyte cytosolic protein
8.744
5q33.1-qter
Promotes T-cell




2 (SH2 domain containing


development and




leukocyte protein of 76 kDa)


activation




(LCP2)


5366
PMAIP1
Phorbol-12-myristate-13-
8.543
18q21.32
Unknown




acetate-induced protein 1




(PMAIP1)


8038
ADAM12
ADAM metallopeptidase
7.605
10q26.3
Involved in cell-cell and




domain 12 (meltrin alpha)


cell-matrix interactions




(ADAM12), transcript variant 1


25878
MXRA5
Matrix-remodeling associated
6.865
Xp22.33
Matrix remodeling




5 (MXRA5)


1123
CHN1
Chimerin (chimaerin) 1
6.857
2q31-q32.1
Rho GTPase activating




(CHN1)


protein


3310
HSPA6
Heat shock 70 kDa protein 6
6.76
1q23
Involved in protein







conformational







interactions


10631
POSTN
Periostin, osteoblast specific
6.732
13q13.3
Promotes integrin-




factor (POSTN)


dependent cell adhesion







and motility, involved in







extracellular matrix







deposition


11211
FZD10
Frizzled homolog 10
6.701
12q24.33
Receptor for the




(Drosophila) (FZD10)


wingless type MMTV







integration site family


27242
TNFRSF21
Tumor necrosis factor
6.649
6p21.1-12.2
Activates NF-kappaB




receptor superfamily, member


and MAPK8/JNK,




21 (TNFRSF21)


induces cell apoptosis,







involved in







inflammation and







immune regulation.


25891
DKFZP586H2123
Regeneration associated
6.199
11p13
Unknown




muscle protease, transcript




variant 2


79084
MEP50
WD repeat domain 77
6.144
1p13.2
Involved in the







methylation and







assembly of







spliceosomal snRNAs







Sm proteins


50509
COL5A3
Collagen, type V, alpha 3
6.118
19p13.2
Extracellular protein,




(COL5A3)


associated with







formation of fibrils, and







some connective tissue







pathology such as







inflammation, cancer







and atherosclerosis


6205
RPS11
Ribosomal protein S11
6.095
19q13.3
Involved in the




(RPS11)


recognition of







termination codons


55803
CENTA2
Centaurin, alpha 2 (CENTA2)
6.09
17q11.2
A







phosphatidylinositide-







binding protein present







in the dense membrane







fractions of cell extracts


90161
HS6ST2
Heparan sulfate 6-O-
0.153
Xq26.2
Plays a role in growth




sulfotransferase 2 (HS6ST2)


factor signaling, cell







adhesion, and enzymatic







catalysis. Maybe







involved in







vascularization by







mediating FGF







signaling


4969
OGN
Osteoglycin (osteoinductive
0.147
9q22
Regulates collagen




factor, mimecan) (OGN),


fibrillogenesis




transcript variant 3


9687
GREB1
GREB1 protein, transcript
0.116
2p25.1
Transcription factor;




variant a


inhibits cell







proliferation


6401
SELE
Selectin E (endothelial
0.112
1q22-q25
Cell surface lycoprotein.




adhesion molecule 1) (SELE)


Inhibits cell adhesion.







Early marker of







inflammation


5350
PLN
Phospholamban
0.108
6q22.1
Inhibits sarcoplasmic







reticulum Ca(2+)-







ATPase activity
















TABLE 5







Genes specifically regulated in tumor endothelium.














Fold difference







Parametric
of geom means



Gene


p-value
(Tumor/Normal)
Probe set
Description
UG cluster
symbol
Map
















p < 1e−07
29.062
206026_s_at
tumor necrosis factor,
Hs.437322
TNFAIP6
2q23.3





alpha-induced protein 6





(TNFAIP6), mRNA.


p < 1e−07
0.274
213803_at
Karyopherin (importin)
Hs.532793
KPNB1
17q21.32





beta 1












p < 1e−07
0.205
235309_at
CDNA clone
Hs.526499
16





IMAGE: 4140029













  1e−007
2.85
228204_at
proteasome (prosome,
Hs.89545
PSMB4
1q21





macropain) subunit, beta





type, 4 (PSMB4), mRNA.


  1e−007
13.144
211597_s_at
homeodomain-only
Hs.121443
HOP
4q11-q12





protein (HOP), transcript





variant 2, mRNA.


  2e−007
3.116
213848_at
Dual specificity
Hs.3843
DUSP7
3p21





phosphatase 7


  3e−007
3.599
233274_at
NCK adaptor protein 1
Hs.477693
NCK1
3q21


  3e−007
0.266
212653_s_at
EH domain binding
Hs.271667
EHBP1
2p15





protein 1 (EHBP1),





mRNA.


  4e−007
10.215
214081_at
plexin domain containing
Hs.125036
PLXDC1
17q21.1





1 (PLXDC1), mRNA.


  4e−007
0.27
213364_s_at
sorting nexin 1 (SNX1),
Hs.188634
SNX1
15q22.31





transcript variant 2,





mRNA.


  4e−007
36.848
219454_at
EGF-like-domain,
Hs.12844
EGFL6
Xp22





multiple 6 (EGFL6),





mRNA.


  4e−007
18.969
213943_at
twist homolog 1
Hs.66744
TWIST1
7p21.2





(acrocephalosyndactyly





3; Saethre-Chotzen





syndrome) (Drosophila)





(TWIST1), mRNA.


  4e−007
0.327
225123_at
Sestrin 3
Hs.120633
SESN3
11q21


  5e−007
0.165
230081_at
phosphatidylinositol-
Hs.145404
PLCXD3
5p13.1





specific phospholipase C,





X domain containing 3





(PLCXD3), mRNA.


  7e−007
5.249
228579_at
Potassium voltage-gated
Hs.374023
KCNQ3
8q24





channel, KQT-like





subfamily, member 3


  8e−007
6.199
213661_at
regeneration associated
Hs.55044
DKFZP586H2123
11p13





muscle protease





(DKFZP586H2123),





transcript variant 2,





mRNA.


  9e−007
6.701
219764_at
frizzled homolog 10
Hs.31664
FZD10
12q24.33





(Drosophila) (FZD10),





mRNA.


  9e−007
3.38
212044_s_at
ribosomal protein L27a
Hs.523463
RPL27A
11p15





(RPL27A), mRNA.


  1e−006
0.42
213574_s_at
Karyopherin (importin)
Hs.532793
KPNB1
17q21.32





beta 1


 1.1e−006
7.684
219700_at
plexin domain containing
Hs.125036
PLXDC1
17q21.1





1 (PLXDC1), mRNA.


 1.2e−006
0.294
222791_at
round spermatid basic
Hs.486285
RSBN1
1p13.2





protein 1 (RSBN1),





mRNA.


 1.2e−006
0.232
221589_s_at
Aldehyde dehydrogenase
Hs.293970
ALDH6A1
14q24.3





6 family, member A1


 1.2e−006
7.724
204285_s_at
phorbol-12-myristate-13-
Hs.96
PMAIP1
18q21.32





acetate-induced protein 1





(PMAIP1), mRNA.


 1.4e−006
10.546
204595_s_at
stanniocalcin 1 (STC1),
Hs.25590
STC1
8p21-p11.2





mRNA.


 1.5e−006
3.695
225147_at
pleckstrin homology,
Hs.487479
PSCD3
7p22.1





Sec7 and coiled-coil





domains 3 (PSCD3),





mRNA.












 1.5e−006
9.368
234723_x_at
CDNA: FLJ21228 fis,
Hs.306716
7





clone COL00739













 1.5e−006
0.374
212498_at
Membrane-associated
Hs.432862
MARCH-
5p15.2





ring finger (C3HC4) 6

VI


 1.5e−006
13.326
230746_s_at
stanniocalcin 1 (STC1),
Hs.25590
STC1
8p21-p11.2





mRNA.


 1.6e−006
6.09
219358_s_at
centaurin, alpha 2
Hs.514063
CENTA2
17q11.2





(CENTA2), mRNA.


 1.8e−006
0.346
201425_at
aldehyde dehydrogenase
Hs.436437
ALDH2
12q24.2





2 family (mitochondrial)





(ALDH2), nuclear gene





encoding mitochondrial





protein, mRNA.


 1.9e−006
4.094
225505_s_at
chromosome 20 open
Hs.29341
C20orf81
20p13





reading frame 81





(C20orf81), mRNA.


 2.1e−006
0.284
219939_s_at
cold shock domain
Hs.69855
CSDE1
1p22





containing E1, RNA-





binding (CSDE1),





transcript variant 2,





mRNA.


 2.2e−006
4.136
221059_s_at
coactosin-like 1
Hs.289092
COTL1
16q24.1





(Dictyostelium)





(COTL1), mRNA.


 2.2e−006
6.649
218856_at
tumor necrosis factor
Hs.443577
TNFRSF21
6p21.1-12.2





receptor superfamily,





member 21 (TNFRSF21),





mRNA.


 2.4e−006
3.826
205068_s_at
Rho GTPase activating
Hs.293593
ARHGAP26
5q31





protein 26 (ARHGAP26),





mRNA.


 2.4e−006
4.807
242100_at
chondroitin sulfate
Hs.213137
CSS3
5q23.3





synthase 3 (CSS3),





mRNA.


 2.4e−006
5.981
206377_at
forkhead box F2
Hs.484423
FOXF2
6p25.3





(FOXF2), mRNA.


 2.4e−006
0.327
234512_x_at
PREDICTED: similar to
Hs.535174
LOC442159
6





Rpl7a protein





(LOC442159), mRNA.


 2.6e−006
4.037
232304_at
Pellino homolog 1
Hs.7886
PELI1
2p13.3





(Drosophila)












 2.7e−006
5.391
242546_at
LOC440156
Hs.529095
14q11.1













 2.7e−006
3.362
227295_at
IKK interacting protein
Hs.252543
IKIP
12q23.1





(IKIP), transcript variant





3.1, mRNA.


 2.8e−006
4.563
1553575_at
Unknown


 2.9e−006
3.761
214924_s_at
OGT(O-Glc-NAc
Hs.535711
OIP106
3p25.3-p24.1





transferase)-interacting





protein 106 KDa





(OIP106), mRNA.


  3e−006
6.144
242398_x_at
WD repeat domain 77
Hs.204773
MEP50
1p13.2


 3.5e−006
8.543
204286_s_at
phorbol-12-myristate-13-
Hs.96
PMAIP1
18q21.32





acetate-induced protein 1





(PMAIP1), mRNA.


 3.7e−006
3.271
241632_x_at
ESTs


 3.9e−006
0.258
209512_at
hydroxysteroid
Hs.59486
HSDL2
9q32





dehydrogenase like 2





(HSDL2), mRNA.


  4e−006
4.384
219359_at
hypothetical protein
Hs.353181
FLJ22635
11p15.5





FLJ22635 (FLJ22635),





mRNA.












  4e−006
0.279
227719_at
CDNA FLJ37828 fis,
Hs.123119
13





clone BRSSN2006575













  4e−006
3.268
226063_at
vav 2 oncogene (VAV2),
Hs.369921
VAV2
9q34.1





mRNA.


  4e−006
8.744
205269_at
lymphocyte cytosolic
Hs.304475
LCP2
5q33.1-qter





protein 2 (SH2 domain





containing leukocyte





protein of 76 kDa)





(LCP2), mRNA.


 4.1e−006
3.906
215599_at
SMA4
Hs.482411
SMA4
5q13












 4.2e−006
0.434
214527_s_at
polyglutamine binding protein 1
PQBP1
Xp11.23





(PQBP1), transcript variant 5, mRNA.













 4.5e−006
4.068
220817_at
transient receptor
Hs.262960
TRPC4
13q13.1-q13.2





potential cation channel,





subfamily C, member 4





(TRPC4), mRNA.


 4.6e−006
0.237
202920_at
ankyrin 2, neuronal
Hs.137367
ANK2
4q25-q27





(ANK2), transcript





variant 2, mRNA.


 4.8e−006
6.865
209596_at
matrix-remodelling
Hs.369422
MXRA5
Xp22.33





associated 5 (MXRA5),





mRNA.


 5.1e−006
0.324
201737_s_at
membrane-associated
Hs.432862
38417
5p15.2





ring finger (C3HC4) 6





(MARCH6), mRNA.


 5.3e−006
0.256
224763_at
ribosomal protein L37
Hs.80545
RPL37
5p13





(RPL37), mRNA.


 5.4e−006
6.857
212624_s_at
chimerin (chimaerin) 1
Hs.380138
CHN1
2q31-q32.1





(CHN1), transcript





variant 2, mRNA.


 5.8e−006
0.203
222486_s_at
ADAM metallopeptidase
Hs.534115
ADAMTS1
21q21.2





with thrombospondin





type 1 motif, 1





(ADAMTS1), mRNA.


 5.9e−006
0.294
202908_at
Wolfram syndrome 1
Hs.518602
WFS1
4p16





(wolframin) (WFS1),





mRNA.












 6.2e−006
0.267
242051_at
Transcribed locus
Hs.130260
X













 6.3e−006
0.376
205412_at
acetyl-Coenzyme A
Hs.232375
ACAT1
11q22.3-q23.1





acetyltransferase 1





(acetoacetyl Coenzyme A





thiolase) (ACAT1),





nuclear gene encoding





mitochondrial protein,





mRNA.










 6.5e−006
7.988
224549_x_at














 6.8e−006
0.253
213272_s_at
promethin (LOC57146),
Hs.258212
LOC57146
16p12





mRNA.


 7.2e−006
4.12
242369_x_at
Nuclear receptor
Hs.446678
NCOA2
8q13.3





coactivator 2


 7.3e−006
0.26
226625_at
Transforming growth
Hs.482390
TGFBR3
1p33-p32





factor, beta receptor III





(betaglycan, 300 kDa)


 7.7e−006
0.245
219511_s_at
synuclein, alpha
Hs.426463
SNCAIP
5q23.1-q23.3





interacting protein





(synphilin) (SNCAIP),





mRNA.


 7.8e−006
4.059
222449_at
transmembrane, prostate
Hs.517155
TMEPAI
20q13.31-q13.33





androgen induced RNA





(TMEPAI), transcript





variant 3, mRNA.


  8e−006
2.237
212351_at
eukaryotic translation
Hs.283551
EIF2B5
3q27.1





initiation factor 2B,





subunit 5 epsilon, 82 kDa





(EIF2B5), mRNA.


 8.1e−006
3.836
219634_at
carbohydrate (chondroitin
Hs.17569
CHST11
12q





4) sulfotransferase 11





(CHST11), mRNA.


 8.3e−006
0.418
226751_at
chromosome 2 open
Hs.212885
C2orf32
2p14





reading frame 32





(C2orf32), mRNA.


 8.3e−006
5.037
209081_s_at
collagen, type XVIII,
Hs.517356
COL18A1
21q22.3





alpha 1 (COL18A1),





transcript variant 2,





mRNA.


  9e−006
2.362
205812_s_at
transmembrane emp24
Hs.279929
TMED9
5q35.3





protein transport domain





containing 9 (TMED9),





mRNA.


 9.3e−006
2.862
234985_at
Hypothetical protein
Hs.205865
LOC143458
11p13





LOC143458


 9.6e−006
3.652
242029_at
Fibronectin type III
Hs.159430
FNDC3B
3q26.31





domain containing 3B


 9.6e−006
0.386
211666_x_at
ribosomal protein L3
Hs.119598
RPL3
22q13





(RPL3), mRNA.


 9.8e−006
4.815
202465_at
procollagen C-
Hs.202097
PCOLCE
7q22





endopeptidase enhancer





(PCOLCE), mRNA.


1.06e−005
0.303
203799_at
CD302 antigen (CD302),
Hs.130014
CD302
2q24.2





mRNA.


1.1e−005
3.787
228253_at
lysyl oxidase-like 3
Hs.469045
LOXL3
2p13





(LOXL3), mRNA.


1.1e−005
3.674
209685_s_at
protein kinase C, beta 1
Hs.460355
PRKCB1
16p11.2





(PRKCB1), transcript





variant 2, mRNA.


1.11e−005
0.33
224445_s_at
zinc finger, FYVE
Hs.549192
ZFYVE21
14q32.33





domain containing 21





(ZFYVE21), mRNA.


1.13e−005
3.466
222379_at
Potassium voltage-gated
Hs.348522
KCNE4
2q36.3





channel, Isk-related





family, member 4


1.15e−005
6.118
52255_s_at
collagen, type V, alpha 3
Hs.235368
COL5A3
19p13.2





(COL5A3), mRNA.


1.17e−005
2.821
233180_at
Ring finger protein 152
Hs.465316
RNF152
18q21.33


1.18e−005
0.232
212224_at
aldehyde dehydrogenase
Hs.76392
ALDH1A1
9q21.13





1 family, member A1





(ALDH1A1), mRNA.


1.2e−005
6.095
213350_at
ribosomal protein S11
Hs.433529
RPS11
19q13.3





(RPS11), mRNA.


1.2e−005
3.729
226911_at
hypothetical protein
Hs.20103
FLJ39155
5p13.2-p13.1





FLJ39155 (FLJ39155),





transcript variant 4,





mRNA.


1.21e−005
2.975
219102_at
reticulocalbin 3, EF-hand
Hs.439184
RCN3
19q13.33





calcium binding domain





(RCN3), mRNA.


1.25e−005
0.115
222722_at
osteoglycin
Hs.109439
OGN
9q22





(osteoinductive factor,





mimecan) (OGN),





transcript variant 3,





mRNA.


1.29e−005
0.17
227703_s_at
synaptotagmin-like 4
Hs.522054
SYTL4
Xq21.33





(granuphilin-a) (SYTL4),





mRNA.


1.3e−005
0.46
211988_at
SWI/SNF related, matrix
Hs.463010
SMARCE1
17q21.2





associated, actin





dependent regulator of





chromatin, subfamily e,





member 1 (SMARCE1),





mRNA.


1.31e−005
0.238
204793_at
G protein-coupled
Hs.522730
GPRASP1
Xq22.1





receptor associated





sorting protein 1





(GPRASP1), mRNA.


1.34e−005
0.339
229891_x_at
KIAA1704
Hs.507922
KIAA1704
13q13-q14


1.35e−005
0.215
228554_at
MRNA; cDNA
Hs.32405

11





DKFZp586G0321 (from





clone DKFZp586G0321)


1.38e−005
0.299
210950_s_at
farnesyl-diphosphate
Hs.546253
FDFT1
8p23.1-p22





farnesyltransferase 1





(FDFT1), mRNA.


1.38e−005
4.593
220014_at
mesenchymal stem cell
Hs.157461
LOC51334
5q23.1





protein DSC54





(LOC51334), mRNA.


 1.4e−005
0.262
225162_at
SH3 domain protein D19
Hs.519018
SH3D19
4q31.3





(SH3D19), mRNA.


1.41e−005
0.387
212408_at
torsin A interacting
Hs.496459
TOR1AIP1
1q24.2





protein 1 (TOR1AIP1),





mRNA.


1.41e−005
0.446
201054_at
heterogeneous nuclear
Hs.96996
HNRPA0
5q31





ribonucleoprotein A0





(HNRPA0), mRNA.


1.47e−005
4.235
204639_at
adenosine deaminase
Hs.407135
ADA
20q12-q13.11





(ADA), mRNA.












 1.5e−005
4.351
225646_at
cathepsin C (CTSC), transcript variant 2,
CTSC
11q14.1-q14.3





mRNA.













1.51e−005
3.657
209969_s_at
signal transducer and
Hs.470943
STAT1
2q32.2





activator of transcription





1, 91 kDa (STAT1),





transcript variant beta,





mRNA.


1.54e−005
2.663
236249_at
IKK interacting protein
Hs.252543
IKIP
12q23.1





(IKIP), transcript variant





1, mRNA.


1.58e−005
3.274
218804_at
transmembrane protein
Hs.503074
TMEM16A
11q13.3





16A (TMEM16A),





mRNA.


1.59e−005
0.427
221988_at
Hypothetical protein
Hs.356467
MGC2747
19p13.11





MGC2747


1.59e−005
2.779
204786_s_at
interferon (alpha, beta
Hs.549042
IFNAR2
21q22.11





and omega) receptor 2





(IFNAR2), transcript





variant 1, mRNA.


1.61e−005
0.413
200023_s_at
eukaryotic translation
Hs.516023
EIF3S5
11p15.4





initiation factor 3, subunit





5 epsilon, 47 kDa





(EIF3S5), mRNA.


1.62e−005
4.278
201596_x_at
keratin 18 (KRT18),
Hs.406013
KRT18
12q13





transcript variant 2,





mRNA.


1.64e−005
0.32
222605_at
REST corepressor 3
Hs.356399
RCOR3
1q32.3





(RCOR3), mRNA.


1.64e−005
0.371
209733_at
Hypothetical protein
Hs.348844
LOC286440
Xq22.3





LOC286440


1.67e−005
4.144
232458_at
Collagen, type III, alpha
Hs.443625
COL3A1
2q31





1 (Ehlers-Danlos





syndrome type IV,





autosomal dominant)


1.7e−005
0.282
224901_at
Stearoyl-CoA desaturase 5
Hs.379191
SCD4
4q21.3


1.73e−005
4.385
233912_x_at
ELMO domain
Hs.450105
ELMOD2
4q31.21





containing 2


1.76e−005
5.792
220301_at
chromosome 18 open
Hs.280781
C18orf14
18q22.1





reading frame 14





(C18orf14), mRNA.


1.76e−005
0.388
207170_s_at
LETM1 domain
Hs.370457
LETMD1
12q13.12





containing 1 (LETMD1),





transcript variant 3,





mRNA.


1.82e−005
0.418
201076_at
NHP2 non-histone
Hs.182255
NHP2L1
22q13.2-q13.31





chromosome protein 2-





like 1 (S. cerevisiae)





(NHP2L1), transcript





variant 2, mRNA.


1.82e−005
0.252
228885_at
MAM domain containing
Hs.127386
MAMDC2
9q21.11





2 (MAMDC2), mRNA.


1.85e−005
0.411
212609_s_at
V-akt murine thymoma
Hs.498292
AKT3
1q43-q44





viral oncogene homolog





3 (protein kinase B,





gamma)


1.86e−005
4.564
221558_s_at
lymphoid enhancer-
Hs.555947
LEF1
4q23-q25





binding factor 1 (LEF1),





mRNA.


1.87e−005
3.054
235204_at
Ectonucleoside
Hs.369424
ENTPD7
10





triphosphate





diphosphohydrolase 7


 1.9e−005
3.283
202820_at
aryl hydrocarbon receptor
Hs.171189
AHR
7p15





(AHR), mRNA.


1.91e−005
4.787
1565823_at
septin 7 (SEPT7),
Hs.191346
38602
7p14.3-p14.1





transcript variant 2





mRNA.


1.93e−005
0.342
221726_at
ribosomal protein L22
Hs.515329
RPL22
1p36.3-p36.2





(RPL22), mRNA.


1.93e−005
0.485
202029_x_at
ribosomal protein L38
Hs.380953
RPL38
17q23-q25





(RPL38), mRNA.


1.94e−005
0.258
209513_s_at
hydroxysteroid
Hs.59486
HSDL2
9q32





dehydrogenase like 2





(HSDL2), mRNA.












1.95e−005
0.356
226806_s_at
MRNA; cDNA
Hs.379253
 1





DKFZp686J23256 (from





clone DKFZp686J23256)


2.01e−005
4.146
1558048_x_at
Unknown


2.03e−005
3.183
237494_at
Transcribed locus
Hs.174934
15













2.09e−005
0.306
201432_at
catalase (CAT), mRNA.
Hs.502302
CAT
11p13


2.11e−005
3.698
203878_s_at
matrix metallopeptidase
Hs.143751
MMP11
22q11.23





11 (stromelysin 3)





(MMP11), mRNA.


2.11e−005
2.481
212323_s_at
vacuolar protein sorting
Hs.439381
VPS13D
1p36.22-p36.21





13D (yeast) (VPS13D),





transcript variant 2,





mRNA.












2.11e−005
0.215
242488_at
CDNA FLJ38396 fis,
Hs.155736
 1





clone FEBRA2007957













214e−005
2.201
219092_s_at
chromosome 9 open
Hs.16603
C9orf12
9q21.33-q22.31





reading frame 12





(C9orf12), mRNA.


215e−005
2.239
217118_s_at
chromosome 22 open
Hs.369682
C22orf9
22q13.31





reading frame 9





(C22orf9), transcript





variant 2, mRNA.


2.29e−005
0.44
214097_at
Ribosomal protein S21
Hs.190968
RPS21
20q13.3


2.29e−005
5.232
225799_at
hypothetical protein
Hs.446688
MGC4677
2p11.2





MGC4677 (MGC4677),





mRNA.


2.3e−005
3.258
244197_x_at
CCR4-NOT transcription
Hs.133350
CNOT2
12q15





complex, subunit 2


2.37e−005
7.605
202952_s_at
ADAM metallopeptidase
Hs.386283
ADAM12
10q26.3





domain 12 (meltrin





alpha) (ADAM12),





transcript variant 1,





mRNA.


 2.4e−005
0.462
201030_x_at
lactate dehydrogenase B
Hs.446149
LDHB
12p12.2-p12.1





(LDHB), mRNA.


2.41e−005
0.349
208643_s_at
X-ray repair
Hs.388739
XRCC5
2q35





complementing defective





repair in Chinese hamster





cells 5 (double-strand-





break rejoining; Ku





autoantigen, 80 kDa)





(XRCC5), mRNA.


2.5e−005
0.381
200013_at
ribosomal protein L24
Hs.477028
RPL24
3q12





(RPL24), mRNA.


2.57e−005
10.355
221731_x_at
chondroitin sulfate
Hs.443681
CSPG2
5q14.3





proteoglycan 2 (versican)





(CSPG2), mRNA.


2.66e−005
0.175
211569_s_at
L-3-hydroxyacyl-
Hs.438289
HADHSC
4q22-q26





Coenzyme A





dehydrogenase, short





chain (HADHSC),





mRNA.


2.69e−005
0.412
201023_at
TAF7 RNA polymerase
Hs.438838
TAF7
5q31





II, TATA box binding





protein (TBP)-associated





factor, 55 kDa (TAF7),





mRNA.


2.71e−005
5.473
224254_x_at
Transferrin
Hs.518267
TF
3q22.1


2.75e−005
4.279
213479_at
neuronal pentraxin II
Hs.3281
NPTX2
7q21.3-q22.1





(NPTX2), mRNA.


2.81e−005
3.34
230440_at
PREDICTED: zinc finger
Hs.54925
ZNF469
16





protein 469 (ZNF469),





mRNA.


2.86e−005
3.995
227347_x_at
hairy and enhancer of
Hs.154029
HES4
1p36.33





split 4 (Drosophila)





(HES4), mRNA.


2.87e−005
2.894
218131_s_at
GATA zinc finger
Hs.118964
GATAD2A
19p13.11





domain containing 2A





(GATAD2A), mRNA.












 2.9e−005
0.327
235612_at
Transcribed locus,
Hs.396796
 1





moderately similar to





NP_858931.1 NFS1





nitrogen fixation 1





isoform b precursor;





cysteine desulfurase;





nitrogen-fixing bacteria





S-like protein; nitrogen





fixation 1 (S. cerevisiae,





homolog) [Homo





sapiens]


2.94e−005
0.186
229308_at
Transcribed locus
Hs.355689
18













3.01e−005
2.617
223617_x_at
ATPase family, AAA
Hs.23413
ATAD3B
1p36.33





domain containing 3B





(ATAD3B), mRNA.


3.01e−005
4.273
240655_at
Activated leukocyte cell
Hs.150693
ALCAM
3q13.1





adhesion molecule


3.07e−005
2.553
209030_s_at
immunoglobulin
Hs.370510
IGSF4
11q23.2





superfamily, member 4





(IGSF4), mRNA.


3.07e−005
2.812
210069_at
carnitine
Hs.439777
CPT1B
22q13.33





palmitoyltransferase 1B





(muscle) (CPT1B),





nuclear gene encoding





mitochondrial protein,





transcript variant 3,





mRNA.


 3.1e−005
2.462
213258_at
Tissue factor pathway
Hs.516578
TFPI
2q31-q32.1





inhibitor (lipoprotein-





associated coagulation





inhibitor)










3.28e−005
4.4
241686_x_at
ESTs, Weakly similar to hypothetical protein FLJ20378 [Homo





sapiens] [H. sapiens]













3.33e−005
5.315
1553186_x_at
RAS and EF-hand
Hs.129136
RASEF
9q21.32





domain containing


3.34e−005
0.361
226280_at
BCL2/adenovirus E1B
Hs.283454
BNIP2
15q22.2





19 kDa interacting protein 2


3.34e−005
0.481
218929_at
collaborates/cooperates
Hs.32922
CARF
4q35.1





with ARF (alternate





reading frame) protein





(CARF), mRNA.


3.36e−005
2.651
61734_at
Reticulocalbin 3, EF-
Hs.439184
RCN3
19q13.33





hand calcium binding





domain


3.38e−005
3.492
204735_at
phosphodiesterase 4A,
Hs.89901
PDE4A
19p13.2





cAMP-specific





(phosphodiesterase E2





dunce homolog,





Drosophila) (PDE4A),





mRNA.


 3.4e−005
4.417
47550_at
leucine zipper, putative
Hs.521432
LZTS1
8p22





tumor suppressor 1





(LZTS1), mRNA.


3.41e−005
0.309
225698_at
TIGA1 (TIGA1), mRNA.
Hs.12082
TIGA1
5q21-q22


3.42e−005
0.304
211986_at
AHNAK nucleoprotein
Hs.502756
AHNAK
11q12.2





(desmoyokin) (AHNAK),





transcript variant 1,





mRNA.


3.43e−005
0.31
225387_at
Tetraspanin 5
Hs.118118
TM4SF9
4q23


3.48e−005
0.442
201600_at
prohibitin 2 (PHB2),
Hs.504620
PHB2
12p13





mRNA.


3.5e−005
0.323
225125_at
transmembrane protein
Hs.110702
TMEM32
Xq26.3





32 (TMEM32), mRNA.


3.52e−005
4.82
206637_at
purinergic receptor P2Y,
Hs.2465
P2RY14
3q21-q25





G-protein coupled, 14





(P2RY14), mRNA.


 3.6e−005
0.475
221725_at
WAS protein family,
Hs.469244
WASF2
1p36.11-p34.3





member 2


3.63e−005
0.387
209385_s_at
proline synthetase co-
Hs.304792
PROSC
8p11.2





transcribed homolog





(bacterial) (PROSC),





mRNA.


3.65e−005
2.247
218018_at
pyridoxal (pyridoxine,
Hs.284491
PDXK
21q22.3





vitamin B6) kinase





(PDXK), mRNA.


3.68e−005
2.36
224598_at
mannosyl (alpha-1,3-)-
Hs.437277
MGAT4B
5q35





glycoprotein beta-1,4-N-





acetylglucosaminyltransferase,





isoenzyme B





(MGAT4B), transcript





variant 1, mRNA.


3.71e−005
0.502
225489_at
transmembrane protein
Hs.43899
TMEM18
2p25.3





18 (TMEM18), mRNA.












3.72e−005
0.264
239262_at
CDNA FLJ26242 fis,
Hs.377660
11





clone DMC00770













3.75e−005
0.277
226184_at
formin-like 2 (FMNL2),
Hs.149566
FMNL2
2q23.3





transcript variant 2,





mRNA.


3.77e−005
0.251
228027_at
G protein-coupled
Hs.348493
GPRASP2
Xq22.1





receptor associated





sorting protein 2





(GPRASP2), transcript





variant 2, mRNA.


3.79e−005
0.257
203803_at
prenylcysteine oxidase 1
Hs.551542
PCYOX1
2p13.3





(PCYOX1), mRNA.


3.79e−005
6.76
213418_at
heat shock 70 kDa protein
Hs.3268
HSPA6
1q23





6 (HSP70B′) (HSPA6),





mRNA.












3.82e−005
2.139
1556242_a_at
Homo sapiens, clone
Hs.547780
 8





IMAGE: 3885623, mRNA













3.84e−005
0.407
209447_at
spectrin repeat
Hs.12967
SYNE1
6q25





containing, nuclear





envelope 1 (SYNE1),





transcript variant alpha,





mRNA.


3.89e−005
0.367
213900_at
chromosome 9 open
Hs.118003
C9orf61
9q13-q21





reading frame 61





(C9orf61), mRNA.


3.91e−005
2.496
205406_s_at
sperm autoantigenic
Hs.286233
SPA17
11q24.2





protein 17 (SPA17),





mRNA.


3.91e−005
2.719
213344_s_at
H2A histone family,
Hs.477879
H2AFX
11q23.2-q23.3





member X (H2AFX),





mRNA.


3.95e−005
0.318
228551_at
Hypothetical protein
Hs.118166
MGC24039
12p11.21





MGC24039


3.96e−005
4.16
235343_at
Hypothetical protein
Hs.96885
FLJ12505
1q32.3





FLJ12505


4.04e−005
5.126
209082_s_at
collagen, type XVIII,
Hs.517356
COL18A1
21q22.3





alpha 1 (COL18A1),





transcript variant 2,





mRNA.












4.15e−005
0.476
230958_s_at
MRNA; cDNA
Hs.379253
 1





DKFZp686J23256 (from





clone DKFZp686J23256)













4.17e−005
0.38
219054_at
hypothetical protein
Hs.13528
FLJ14054
5p13.3





FLJ14054 (FLJ14054),





mRNA.


 4.2e−005
0.358
208951_at
aldehyde dehydrogenase
Hs.483239
ALDH7A1
5q31





7 family, member A1





(ALDH7A1), mRNA.


 4.2e−005
4.533
205241_at
SCO cytochrome oxidase
Hs.549099
SCO2
22q13.33





deficient homolog 2





(yeast) (SCO2), nuclear





gene encoding





mitochondrial protein,





mRNA.


4.23e−005
0.434
218528_s_at
ring finger protein 38
Hs.333503
RNF38
9p13-p12





(RNF38), transcript





variant 4, mRNA.


4.28e−005
0.489
212131_at
family with sequence
Hs.407368
FAM61A
19q13.11





similarity 61, member A





(FAM61A), mRNA.


4.45e−005
0.365
225546_at
Eukaryotic elongation
Hs.549151
EEF2K
16p12.1





factor-2 kinase


4.45e−005
0.237
229145_at
chromosome 10 open
Hs.426296
C10orf104
10q22.1





reading frame 104





(C10orf104), mRNA.












4.47e−005
0.387
227273_at
Transcribed locus
Hs.483955
10













4.48e−005
3.428
220575_at
hypothetical protein
Hs.287456
FLJ11800
17p11.2





FLJ11800 (FLJ11800),





mRNA.


4.49e−005
0.379
202073_at
optineurin (OPTN),
Hs.332706
OPTN
10p13





transcript variant 2,





mRNA.


4.54e−005
4.845
1559436_x_at
Arrestin, beta 2
Hs.435811
ARRB2
17p13


4.54e−005
4.79
220232_at
stearoyl-CoA desaturase
Hs.379191
SCD5
4q21.3





5 (SCD5), mRNA.












4.59e−005
5.647
233330_s_at
Similar to Ribosome
Hs.455494
9q13





biogenesis protein BMS1





homolog













4.66e−005
3.575
1559410_at
Unknown





4.67e−005
0.425
211769_x_at
tumor differentially
Hs.272168
TDE1
20q13.1-13.3





expressed 1 (TDE1),





transcript variant 1,





mRNA.


4.85e−005
0.443
226529_at
hypothetical protein
Hs.396358
FLJ11273
7p21.3





FLJ11273 (FLJ11273),





mRNA.


4.85e−005
0.413
208697_s_at
eukaryotic translation
Hs.405590
EIF3S6
8q22-q23





initiation factor 3, subunit





6 48 kDa (EIF3S6),





mRNA.


4.85e−005
0.404
225050_at
zinc finger protein 512
Hs.529178
ZNF512
2p23





(ZNF512), mRNA.


 4.9e−005
0.277
208704_x_at
amyloid beta (A4)
Hs.370247
APLP2
11q24





precursor-like protein 2





(APLP2), mRNA.


5.08e−005
0.501
202502_at
acyl-Coenzyme A
Hs.445040
ACADM
1p31





dehydrogenase, C-4 to C-





12 straight chain





(ACADM), nuclear gene





encoding mitochondrial





protein, mRNA.


 5.1e−005
2.907
223276_at
putative small membrane
Hs.29444
NID67
5q33.1





protein NID67 (NID67),





mRNA.


 5.1e−005
0.484
208873_s_at
chromosome 5 open
Hs.429608
C5orf18
5q22-q23





reading frame 18





(C5orf18), mRNA.


5.11e−005
2.383
46665_at
sema domain,
Hs.516220
SEMA4C
2q11.2





immunoglobulin domain





(Ig), transmembrane





domain (TM) and short





cytoplasmic domain,





(semaphorin) 4C





(SEMA4C), mRNA.


5.16e−005
3
1559060_a_at
KIAA1961 gene
Hs.483329
KIAA1961
5q23.3


5.29e−005
2.423
215577_at
Ubiquitin-conjugating
Hs.164853
UBE2E1
3p24.2





enzyme E2E 1 (UBC4/5





homolog, yeast)


5.29e−005
4.015
222252_x_at
leucine rich repeat
Hs.317243
LRRC51
11q13.4





containing 51 (LRRC51),





mRNA.


5.35e−005
0.284
208248_x_at
amyloid beta (A4)
Hs.370247
APLP2
11q24





precursor-like protein 2





(APLP2), mRNA.












5.35e−005
3.191
1558836_at
MRNA; cDNA
Hs.157344
 2





DKFZp667A182 (from





clone DKFZp667A182)













5.35e−005
0.538
209066_x_at
ubiquinol-cytochrome c
Hs.131255
UQCRB
8q22





reductase binding protein





(UQCRB), mRNA.


5.39e−005
4.851
226777_at
A disintegrin and
Hs.386283
ADAM12
10q26.3





metalloproteinase domain





12 (meltrin alpha)


 5.4e−005
0.459
208990_s_at
heterogeneous nuclear
Hs.499891
HNRPH3
10q22





ribonucleoprotein H3





(2H9) (HNRPH3),





transcript variant 2H9A,





mRNA.












5.42e−005
3.146
235122_at
CDNA clone
Hs.403972
 1





IMAGE: 6254031


5.44e−005
2.185
59433_at
Transcribed locus
Hs.416792
X













5.44e−005
0.473
225811_at
Transcribed locus,
Hs.78050

11





weakly similar to





XP_510104.1





PREDICTED: similar to





hypothetical protein





FLJ25224 [Pan





troglodytes]


5.53e−005
0.39
200760_s_at
ADP-ribosylation-like
Hs.518060
ARL6IP5
3p14





factor 6 interacting





protein 5 (ARL6IP5),





mRNA.


5.55e−005
4.287
219025_at
CD248 antigen,
Hs.195727
CD248
11q13





endosialin (CD248),





mRNA.


5.65e−005
3.458
211673_s_at
Molybdenum cofactor
Hs.357128
MOCS1
6p21.3





synthesis 1


5.75e−005
2.33
225947_at
myosin head domain
Hs.302051
MYOHD1
17q12





containing 1 (MYOHD1),





mRNA.


5.78e−005
0.468
225332_at
Keratin associated protein
Hs.549512
KRTAP4-7
17q12-q21





4-7


5.79e−005
3.761
226933_s_at
inhibitor of DNA binding
Hs.519601
ID4
6p22.p21





4, dominant negative





helix-loop-helix protein





(ID4), mRNA.


5.83e−005
0.421
200937_s_at
ribosomal protein L5
Hs.532359
RPL5
1p22.1





(RPL5), mRNA.


5.92e−005
4.185
219263_at
ring finger protein 128
Hs.496542
RNF128
Xq22.3





(RNF128), transcript





variant 2, mRNA.


5.98e−005
2.652
224967_at
UDP-glucose ceramide
Hs.304249
UGCG
9q31





glucosyltransferase


5.98e−005
2.885
222968_at
chromosome 6 open
Hs.109798
C6orf48
6p21.3





reading frame 48





(C6orf48), mRNA.












6.02e−005
4.748
238673_at
Transcribed locus
Hs.359393
 8













6.02e−005
0.418
223306_at
emopamil binding
Hs.433278
EBPL
13q12-q13





protein-like (EBPL),





mRNA.












6.09e−005
2.563
238327_at
Similar to MGC52679
Hs.531314
22q13.33





protein













6.11e−005
0.341
227728_at
Protein phosphatase 1A
Hs.130036
PPM1A
14q23.1





(formerly 2C),





magnesium-dependent,





alpha isoform


6.17e−005
0.212
205466_s_at
heparan sulfate
Hs.507348
HS3ST1
4p16





(glucosamine) 3-O-





sulfotransferase 1





(HS3ST1), mRNA.


6.24e−005
0.452
202512_s_at
APG5 autophagy 5-like
Hs.486063
APG5L
6q21





(S. cerevisiae) (APG5L),





mRNA.


6.25e−005
2.159
202297_s_at
RER1 retention in
Hs.525527
RER1
1pter-q24





endoplasmic reticulum 1





homolog (S. cerevisiae)





(RER1), mRNA.


6.29e−005
5.165
242862_x_at
ESTs


6.34e−005
0.41
234339_s_at
glioma tumor suppressor
Hs.421907
GLTSCR2
19q13.3





candidate region gene 2





(GLTSCR2), mRNA.


6.38e−005
3.706
215588_x_at
RIO kinase 3 (yeast)
Hs.445511
RIOK3
18q11.2


6.45e−005
0.446
202378_s_at
leptin receptor
Hs.23581
LEPROT
1p31.2





overlapping transcript





(LEPROT), mRNA.












6.52e−005
4.272
234675_x_at
CDNA: FLJ23566 fis,
Hs.532596
14





clone LNG10880













6.61e−005
2.48
200734_s_at
ADP-ribosylation factor 3
Hs.119177
ARF3
12q13





(ARF3), mRNA.


6.64e−005
0.373
202630_at
amyloid beta precursor
Hs.84084
APPBP2
17q21-q23





protein (cytoplasmic tail)





binding protein 2





(APPBP2), mRNA.


6.64e−005
0.417
212549_at
signal transducer and
Hs.132864
STAT5B
17q11.2





activator of transcription





5B (STAT5B), mRNA.


6.66e−005
0.332
235072_s_at
Transcribed locus
Hs.94499

 6


6.72e−005
0.441
201535_at
ubiquitin-like 3 (UBL3),
Hs.145575
UBL3
13q12-q13





mRNA.


6.79e−005
2.242
224612_s_at
DnaJ (Hsp40) homolog,
Hs.164419
DNAJC5
20q13.33





subfamily C, member 5


 6.8e−005
4.528
215179_x_at
Placental growth factor,
Hs.252820
PGF
14q24-q31





vascular endothelial





growth factor-related





protein


6.81e−005
0.361
218191_s_at
LMBR1 domain
Hs.271643
LMBRD1
6q13





containing 1 (LMBRD1),





mRNA.


6.82e−005
3.714
206792_x_at
phosphodiesterase 4C,
Hs.437211
PDE4C
19p13.11





cAMP-specific





(phosphodiesterase E1





dunce homolog,





Drosophila) (PDE4C),





mRNA.


6.85e−005
2.964
44783_s_at
hairy/enhancer-of-split
Hs.234434
HEY1
8q21





related with YRPW motif





1 (HEY1), mRNA.


7.02e−005
0.354
211942_x_at
Ribosomal protein L13a
Hs.546356
RPL13A
19q13.3










7.05e−005
3.112
222358_x_at
ESTs, Weakly similar to hypothetical protein FLJ20378 [Homo





sapiens] [H. sapiens]













7.05e−005
0.387
203427_at
ASF1 anti-silencing
Hs.292316
ASF1A
6q22.31





function 1 homolog A (S. cerevisiae)





(ASF1A),





mRNA.










7.12e−005
5.701
243147_x_at
ESTs, Weakly similar to RMS1_HUMAN REGULATOR OF





MITOTIC SPINDLE ASSEMBLY 1 [H. sapiens]













7.15e−005
5.24
1554334_a_at
DnaJ (Hsp40) homolog,
Hs.513053
DNAJA4
15q25.1





subfamily A, member 4





(DNAJA4), mRNA.


7.18e−005
3.411
204136_at
collagen, type VII, alpha
Hs.476218
COL7A1
3p21.1





1 (epidermolysis bullosa,





dystrophic, dominant and





recessive) (COL7A1),





mRNA.


7.19e−005
0.308
200883_at
ubiquinol-cytochrome c
Hs.528803
UQCRC2
16p12





reductase core protein II





(UQCRC2), mRNA.










7.21e−005
2.399
243249_at
ESTs, Weakly similar to hypothetical protein FLJ20378 [Homo





sapiens] [H. sapiens]













7.25e−005
0.412
218167_at
archaemetzincins-2
Hs.268122
AMZ2
17q24.2





(AMZ2), mRNA.












7.26e−005
4.893
234578_at
MRNA; cDNA
Hs.537604
 1





DKFZp434E1812 (from





clone DKFZp434E1812)













7.26e−005
2.797
203349_s_at
ets variant gene 5 (ets-
Hs.43697
ETV5
3q28





related molecule)





(ETV5), mRNA.


7.32e−005
2.605
212809_at
nuclear factor of
Hs.513470
NFATC2IP
16p11.2





activated T-cells,





cytoplasmic, calcineurin-





dependent 2 interacting





protein (NFATC2IP),





mRNA.


7.34e−005
0.32
230793_at
leucine rich repeat
Hs.116470
LRRC16
6p22.2





containing 16 (LRRC16),





mRNA.


7.35e−005
0.448
203897_at
hypothetical protein A-
Hs.185489
LOC57149
16p11.2





211C6.1 (LOC57149),





mRNA.


7.37e−005
3.381
241718_x_at
ESTs


 7.4e−005
0.451
208740_at
sin3-associated
Hs.524899
SAP18
13q12.11





polypeptide, 18 kDa





(SAP18), mRNA.


7.41e−005
0.392
211749_s_at
vesicle-associated
Hs.66708
VAMP3
1p36.23





membrane protein 3





(cellubrevin) (VAMP3),





mRNA.


7.44e−005
4.754
209360_s_at
runt-related transcription
Hs.149261
RUNX1
21q22.3





factor 1 (acute myeloid





leukemia 1; aml1





oncogene) (RUNX1),





transcript variant 1,





mRNA.


7.45e−005
0.461
225498_at
chromatin modifying
Hs.472471
CHMP4B
20q11.22





protein 4B (CHMP4B),





mRNA.


7.51e−005
4.374
213790_at
A disintegrin and
Hs.386283
ADAM12
10q26.3





metalloproteinase domain





12 (meltrin alpha)


7.57e−005
2.898
230270_at
ESTs


7.64e−005
0.321
219023_at
chromosome 4 open
Hs.435991
C4orf16
4q25





reading frame 16





(C4orf16), mRNA.


7.65e−005
0.116
205862_at
GREB1 protein
Hs.467733
GREB1
2p25.1





(GREB1), transcript





variant a, mRNA.










7.86e−005
4.771
217679_x_at
ESTs, Weakly similar to hypothetical protein FLJ20489 [Homo





sapiens] [H. sapiens]













7.88e−005
2.558
204387_x_at
mitochondrial ribosomal
Hs.458367
MRP63
13p11.1-q11





protein 63 (MRP63),





nuclear gene encoding





mitochondrial protein,





mRNA.


7.89e−005
0.454
226020_s_at
OMA1 homolog, zinc
Hs.425769
OMA1
1p32.2-p32.1





metallopeptidase (S. cerevisiae)





(OMA1),





mRNA.


7.97e−005
2.379
214316_x_at
Calreticulin
Hs.515162
CALR
19p13.3-p13.2


7.99e−005
0.39
218831_s_at
Fc fragment of IgG,
Hs.111903
FCGRT
19q13.3





receptor, transporter,





alpha (FCGRT), mRNA.












8.09e−005
3.577
208246_x_at
hypothetical protein
FLJ20006
16q23.1





FLJ20006













8.13e−005
3.611
231825_x_at
Activating transcription
Hs.546406
ATF7IP
12p13.1





factor 7 interacting





protein


8.16e−005
2.701
236251_at
Integrin, alpha V
Hs.436873
ITGAV
2q31-q32





(vitronectin receptor,





alpha polypeptide,





antigen CD51)


8.18e−005
3.162
232617_at
cathepsin S (CTSS),
Hs.181301
CTSS
1q21





mRNA.


8.21e−005
2.949
31874_at
Growth arrest-specific 2
Hs.322852
GAS2L1
22q12.2





like 1


8.32e−005
4.254
1566887_x_at
KIAA0284
Hs.533721
KIAA0284
14q32.33


8.43e−005
0.475
226297_at
ESTs


8.47e−005
0.458
227293_at
Ligand of numb-protein X
Hs.407755
LNX
4q12


8.47e−005
0.337
227530_at
A kinase (PRKA) anchor
Hs.371240
AKAP12
6q24-q25





protein (gravin) 12


8.52e−005
0.178
211276_at
transcription elongation
Hs.401835
TCEAL2
Xq22.1-q22.3





factor A (SII)-like 2





(TCEAL2), mRNA.


8.57e−005
0.489
208635_x_at
nascent-polypeptide-
Hs.505735
NACA
12q23-q24.1





associated complex alpha





polypeptide (NACA),





mRNA.


8.57e−005
0.416
225574_at
hypothetical protein
Hs.133337
MGC10198
4q35.1





MGC10198





(MGC10198), mRNA.


8.66e−005
3.115
244457_at
Inositol 1,4,5-
Hs.512235
ITPR2
12p11





triphosphate receptor,





type 2


8.72e−005
0.373
226117_at
TRAF-interacting protein
Hs.310640
TIFA
4q25





with a forkhead-





associated domain





(TIFA), mRNA.


8.75e−005
4.112
234762_x_at
Neurolysin
Hs.247460
NLN
5q12.3





(metallopeptidase M3





family)


8.75e−005
2.496
232254_at
F-box protein 25
Hs.438454
FBXO25
8p23.3












8.83e−005
3.223
1570061_at
CDNA clone
Hs.372904
3





IMAGE: 4555030













8.86e−005
0.372
220327_at
vestigial-like (VGL-3),
Hs.435013
VGL-3
3p12.1





mRNA.


8.88e−005
0.501
225326_at
PREDICTED: RNA
Hs.61441
RBM27
 5





binding motif protein 27





(RBM27), mRNA.


8.91e−005
0.347
215294_s_at
SWI/SNF related, matrix
Hs.152292
SMARCA1
Xq25





associated, actin





dependent regulator of





chromatin, subfamily a,





member 1 (SMARCA1),





transcript variant 2,





mRNA.


8.94e−005
3.111
242329_at
PREDICTED:
Hs.437075
LOC401317
 7





hypothetical LOC401317





(LOC401317), mRNA.


8.97e−005
0.37
238613_at
sterile alpha motif and
Hs.444451
ZAK
2q24.2





leucine zipper containing





kinase AZK (ZAK),





transcript variant 2,





mRNA.


8.97e−005
2.137
203459_s_at
vacuolar protein sorting
Hs.269577
VPS16
20p13-p12





16 (yeast) (VPS16),





transcript variant 2,





mRNA.


9.04e−005
5.612
215978_x_at
ATP-binding cassette,
Hs.478916
LOC152719
4p16.3





sub-family A (ABC1),





member 11 (pseudogene)


9.1e−005
0.419
222488_s_at
dynactin 4 (p62)
Hs.328865
DCTN4
5q31-q32





(DCTN4), mRNA.


9.25e−005
0.449
217915_s_at
chromosome 15 open
Hs.274772
C15orf15
15q21





reading frame 15





(C15orf15), mRNA.


9.26e−005
3.027
204184_s_at
adrenergic, beta, receptor
Hs.517493
ADRBK2
22q12.1





kinase 2 (ADRBK2),





mRNA.


9.35e−005
0.152
223395_at
ABI gene family,
Hs.477015
ABI3BP
3q12





member 3 (NESH)





binding protein





(ABI3BP), mRNA.


9.38e−005
3.287
206247_at
MHC class I polypeptide-
Hs.211580
MICB
6p21.3





related sequence B





(MICB), mRNA.


9.41e−005
0.341
222975_s_at
cold shock domain
Hs.69855
CSDE1
1p22





containing E1, RNA-





binding (CSDE1),





transcript variant 2,





mRNA.


9.42e−005
0.43
227407_at
hypothetical protein
Hs.479223
FLJ90013
4p15.32





FLJ90013 (FLJ90013),





mRNA.


9.46e−005
0.424
223189_x_at
myeloid/lymphoid or
Hs.369356
MLL5
7q22.1





mixed-lineage leukemia 5





(trithorax homolog,





Drosophila) (MLL5),





mRNA.


9.63e−005
0.474
200735_x_at
nascent-polypeptide-
Hs.505735
NACA
12q23-q24.1





associated complex alpha





polypeptide (NACA),





mRNA.


9.73e−005
2.027
219099_at
chromosome 12 open
Hs.504545
C12orf5
12p13.3





reading frame 5





(C12orf5), mRNA.


9.74e−005
2.083
243_g_at
microtubule-associated
Hs.517949
MAP4
3p21





protein 4 (MAP4),





transcript variant 2,





mRNA.


9.85e−005
4.389
234981_x_at
Similar to mouse
Hs.192586
LOC134147
5p15.2





2310016A09Rik gene


9.87e−005
0.544
208756_at
eukaryotic translation
Hs.530096
EIF3S2
1p34.1





initiation factor 3, subunit





2 beta, 36 kDa (EIF3S2),





mRNA.










0.0001001
3.738
243442_x_at
ESTs, Weakly similar to ALU1_HUMAN ALU SUBFAMILY J





SEQUENCE CONTAMINATION WARNING ENTRY [H. sapiens]













0.0001002
4.754
223672_at
SH3-domain GRB2-like
Hs.132121
SGIP1
1p31.2





(endophilin) interacting





protein 1 (SGIP1),





mRNA.


0.0001004
3.899
230077_at
Transferrin receptor (p90,
Hs.529618
TFRC
3q29





CD71)


0.0001021
0.362
212215_at
prolyl endopeptidase-like
Hs.112916
PREPL
2p22.1





(PREPL), mRNA.


0.0001021
0.507
225098_at
Abl interactor 2
Hs.471156
ABI2
2q33


0.0001023
0.556
218142_s_at
cereblon (CRBN),
Hs.18925
CRBN
3p26.2





mRNA.


0.0001027
0.445
214177_s_at
pre-B-cell leukemia
Hs.505806
PBXIP1
1q22





transcription factor





interacting protein 1





(PBXIP1), mRNA.


0.0001028
0.227
208791_at
clusterin (complement
Hs.436657
CLU
8p21-p12





lysis inhibitor, SP-40,40,





sulfated glycoprotein 2,





testosterone-repressed





prostate message 2,





apolipoprotein J) (CLU),





transcript variant 1,





mRNA.


0.0001035
2.522
200021_at
cofilin 1 (non-muscle)
Hs.170622
CFL1
11q13





(CFL1), mRNA.


0.0001036
3.432
229801_at
chromosome 10 open
Hs.435775
C10orf47
10p14





reading frame 47





(C10orf47), mRNA.


0.000105
0.303
212731_at
ankyrin repeat domain 46
Hs.530199
ANKRD46
8q22.3





(ANKRD46), mRNA.












0.0001078
0.389
224841_x_at
PREDICTED: RNA, U47 small nuclear
RNU47
 1





(RNU47), misc RNA.












0.0001087
0.419
228905_at
Transcribed locus,
Hs.554337
 8





moderately similar to





XP_517655.1





PREDICTED: similar to





KIAA0825 protein [Pan





troglodytes]













0.0001115
0.379
202314_at
cytochrome P450, family
Hs.417077
CYP51A1
7q21.2-q21.3





51, subfamily A,





polypeptide 1





(CYP51A1), mRNA.


0.000112
0.169
200965_s_at
actin binding LIM protein
Hs.438236
ABLIM1
10q25





1 (ABLIM1), transcript





variant 4, mRNA.


0.0001134
2.869
227850_x_at
CDC42 effector protein
Hs.415791
CDC42EP5
19q13.42





(Rho GTPase binding) 5





(CDC42EP5), mRNA.


0.0001136
0.451
211710_x_at
ribosomal protein L4
Hs.432898
RPL4
15q22





(RPL4), mRNA.










0.000114
4.037
1562062_at
Homo sapiens transcribed sequence with weak similarity to protein





ref: NP_055301.1 (H. sapiens) neuronal thread protein [Homo sapiens]













0.0001144
0.403
224741_x_at
Growth arrest-specific 5
Hs.531856
GAS5
1q23.3


0.0001149
0.503
224689_at
mannosidase, beta A,
Hs.6126
MANBAL
20q11.23-q12





lysosomal-like





(MANBAL), transcript





variant 2, mRNA.


0.0001159
0.449
201154_x_at
ribosomal protein L4
Hs.432898
RPL4
15q22





(RPL4), mRNA.


0.0001171
0.243
202068_s_at
low density lipoprotein
Hs.213289
LDLR
19p13.3





receptor (familial





hypercholesterolemia)





(LDLR), mRNA.


0.0001172
0.438
226541_at
F-box protein 30
Hs.421095
FBXO30
6q24





(FBXO30), mRNA.


0.0001174
2.25
229520_s_at
Chromosome 14 open
Hs.410231
C14orf118
14q22.1-q24.3





reading frame 118


0.0001175
0.224
208792_s_at
clusterin (complement
Hs.436657
CLU
8p21-p12





lysis inhibitor, SP-40,40,





sulfated glycoprotein 2,





testosterone-repressed





prostate message 2,





apolipoprotein J) (CLU),





transcript variant 1,





mRNA.


0.0001184
0.438
212644_s_at
chromosome 14 open
Hs.437831
C14orf32
14q22.2-q22.3





reading frame 32





(C14orf32), mRNA.


0.0001189
0.207
212094_at
PREDICTED: paternally
Hs.147492
PEG10
 7





expressed 10 (PEG10),





mRNA.


0.0001207
7.544
204597_x_at
stanniocalcin 1 (STC1),
Hs.25590
STC1
8p21-p11.2





mRNA.


0.000121
0.518
201696_at
splicing factor,
Hs.469970
SFRS4
1p35.3





arginine/serine-rich 4





(SFRS4), mRNA.


0.0001224
3.248
231411_at
lipoma HMGIC fusion
Hs.507798
LHFP
13q12





partner (LHFP), mRNA.


0.0001224
0.45
203494_s_at
translokin (PIG8),
Hs.101014
PIG8
11q21





mRNA.


0.0001227
0.391
225243_s_at
sarcolemma associated
Hs.476432
SLMAP
3p21.2-p14.3





protein (SLMAP),





mRNA.


0.0001234
2.695
203505_at
ATP-binding cassette,
Hs.429294
ABCA1
9q31.1





sub-family A (ABC1),





member 1












0.000125
2.866
213146_at
KIAA0346 protein
KIAA0346
17p13.1













0.0001252
3.538
235205_at
PREDICTED: similar to
Hs.127286
LOC346887
 8





solute carrier family 16





(monocarboxylic acid





transporters), member 14





(LOC346887), mRNA.












0.0001256
0.494
211994_at
Transcribed locus,
Hs.524171
12





strongly similar to





XP_508919.1





PREDICTED: similar to





protein kinase, lysine





deficient 1; kinase





deficient protein [Pan





troglodytes]













0.0001261
2.214
213836_s_at
WD40 repeat protein
Hs.463964
WIPI49
17q24.2





Interacting with





phosphoInositides of





49 kDa (WIPI49), mRNA.


0.0001271
0.372
212037_at
Pinin, desmosome
Hs.409965
PNN
14q21.1





associated protein












0.000128
2.586
227384_s_at
Similar to KIAA0454
Hs.429365
1q21.1





protein













0.000128
4.614
1553185_at
RAS and EF-hand
Hs.129136
RASEF
9q21.32





domain containing


0.000129
4.274
231183_s_at
jagged 1 (Alagille
Hs.224012
JAG1
20p12.1-p11.23





syndrome) (JAG1),





mRNA.


0.0001299
0.482
222533_at
cereblon (CRBN),
Hs.18925
CRBN
3p26.2





mRNA.


0.0001301
2.543
226695_at
paired related homeobox
Hs.283416
PRRX1
1q24





1 (PRRX1), transcript





variant pmx-1b, mRNA.










0.0001301
3.433
217713_x_at
ESTs, Weakly similar to ALU6_HUMAN ALU SUBFAMILY SP





SEQUENCE CONTAMINATION WARNING ENTRY [H. sapiens]













0.0001306
0.497
225132_at
F-box and leucine-rich
Hs.508284
FBXL3
13q22





repeat protein 3





(FBXL3), mRNA.


0.0001313
0.484
225179_at
Huntingtin interacting
Hs.50308
HIP2
4p14





protein 2


0.0001321
3.609
1557432_at
RAS protein activator
Hs.555904
RASAL2
1q24





like 2


0.0001328
0.224
209612_s_at
alcohol dehydrogenase IB
Hs.4
ADH1B
4q21-q23





(class I), beta polypeptide





(ADH1B), mRNA.


0.0001335
2.071
222753_s_at
signal peptidase complex
Hs.42194
SPCS3
4q34.2





subunit 3 homolog (S. cerevisiae)





(SPCS3),





mRNA.












0.0001337
3.162
1555241_at
Hypothetical gene
Hs.443072
8q21.2





supported by BC055092













0.0001338
0.357
225939_at
Eukaryotic translation
Hs.476782
EIF4E3
3p14





initiation factor 4E





member 3


0.0001341
0.475
217795_s_at
transmembrane protein
Hs.517817
TMEM43
3p25.1





43 (TMEM43), mRNA.


0.0001341
0.442
200920_s_at
B-cell translocation gene
Hs.255935
BTG1
12q22





1, anti-proliferative





(BTG1), mRNA.


0.0001345
0.108
228202_at
Phospholamban
Hs.170839
PLN
6q22.1


0.0001347
2.158
220242_x_at
zinc finger protein 701
Hs.412951
ZNF701
19q13.41





(ZNF701), mRNA.


0.0001348
0.507
201871_s_at
ORF (LOC51035),
Hs.351296
LOC51035
11q12.3





mRNA.


0.0001374
3.17
55583_at
dedicator of cytokinesis 6
Hs.465918
DOCK6
19p13.2





(DOCK6), mRNA.


0.0001375
2.507
225967_s_at
PREDICTED:
Hs.356545
LOC284184
17





hypothetical LOC284184





(LOC284184), mRNA.


0.0001389
0.374
218158_s_at
adaptor protein
Hs.555928
APPL
3p21.1-p14.3





containing pH domain,





PTB domain and leucine





zipper motif 1 (APPL),





mRNA.


0.0001391
2.647
1553569_at
Unknown


0.0001405
2.672
232952_at
HSPC054 protein
Hs.106015
DDEF1
8q24.1-q24.2


0.0001406
3.884
203549_s_at
lipoprotein lipase (LPL),
Hs.180878
LPL
8p22





mRNA.


0.0001436
0.369
226120_at
tetratricopeptide repeat
Hs.303055
TTC8
14q31.3





domain 8 (TTC8),





transcript variant 3,





mRNA.


0.0001439
0.436
222212_s_at
LAG1 longevity
Hs.285976
LASS2
1q21.2





assurance homolog 2 (S. cerevisiae)





(LASS2),





transcript variant 3,





mRNA.


0.0001441
0.45
224755_at
SM-11044 binding
Hs.500674
SMBP
10q24.1





protein


0.0001441
0.409
221588_x_at
aldehyde dehydrogenase
Hs.293970
ALDH6A1
14q24.3





6 family, member A1





(ALDH6A1), nuclear





gene encoding





mitochondrial protein,





mRNA.












0.0001443
0.533
207769_s_at
polyglutamine binding protein 1
PQBP1
Xp11.23





(PQBP1), transcript variant 5, mRNA.













0.0001456
0.496
226336_at
Peptidylprolyl isomerase
Hs.356331
PPIA
7p13-p11.2





A (cyclophilin A)


0.0001456
3.669
216187_x_at
X-ray repair
Hs.549075
XRCC3
14q32.3





complementing defective





repair in Chinese hamster





cells 3


0.000146
2.047
218113_at
transmembrane protein 2
Hs.494146
TMEM2
9q13-q21





(TMEM2), mRNA.


0.0001463
3.512
207598_x_at
X-ray repair
Hs.129727
XRCC2
7q36.1





complementing defective





repair in Chinese hamster





cells 2 (XRCC2), mRNA.


0.0001465
4.648
223697_x_at
chromosome 9 open
Hs.208914
C9orf64
9q21.32





reading frame 64





(C9orf64), mRNA.


0.0001476
2.584
227396_at
Homo sapiens, clone
Hs.374451

11





IMAGE: 4454331, mRNA










0.0001482
2.94
243915_at
ESTs, Weakly similar to 2109260A B cell growth factor [H. sapiens]













0.0001487
2.694
205367_at
adaptor protein with
Hs.489448
APS
7q22





pleckstrin homology and





src homology 2 domains





(APS), mRNA.


0.0001491
0.447
229119_s_at
Hypothetical protein
Hs.462316
TTC19
17p12





LOC125150


0.0001495
0.332
214359_s_at
heat shock 90 kDa protein
Hs.509736
HSPCB
6p12





1, beta (HSPCB), mRNA.












0.0001503
0.185
205381_at
leucine rich repeat containing 17
LRRC17
7q22.1





(LRRC17), transcript variant 1, mRNA.













0.0001503
0.482
213027_at
TROVE domain family,
Hs.288178
SSA2
1q31





member 2


0.0001511
0.348
224734_at
High-mobility group box 1
Hs.434102
HMGB1
13q12


0.0001511
0.474
207974_s_at
S-phase kinase-associated
Hs.171626
SKP1A
5q31





protein 1A (p19A)





(SKP1A), transcript





variant 2, mRNA.












0.0001513
4.07
227952_at
Full length insert cDNA
Hs.355711
 4





clone YI46G04













0.0001514
2.32
240795_at
CDNA clone
Hs.19452

 5





IMAGE: 5288566


0.0001521
0.411
229319_at
Homo sapiens, clone
Hs.33519

 6





IMAGE: 4105966, mRNA


0.0001527
2.413
212414_s_at
septin 6 (SEPT6),
Hs.496666
38601
Xq24





transcript variant II,





mRNA.


0.0001535
0.424
201376_s_at
heterogeneous nuclear
Hs.808
HNRPF
10q11.21-q11.22





ribonucleoprotein F





(HNRPF), mRNA.


0.000154
2.059
50376_at
zinc finger protein 444
Hs.24545
ZNF444
19q13.43





(ZNF444), mRNA.


0.0001543
3.085
233319_x_at
Phosphatase and actin
Hs.225641
PHACTR4
1p35.3





regulator 4


0.000155
0.507
221689_s_at
Down syndrome critical
Hs.408790
DSCR5
21q22.2





region gene 5 (DSCR5),





transcript variant 2,





mRNA.


0.000156
2.319
229200_at
Hypothetical LOC400813
Hs.13742

1q44


0.0001562
4.302
237475_x_at
Selenoprotein P, plasma, 1
Hs.275775
SEPP1
5q31


0.0001564
2.541
1560817_at
Mov10, Moloney
Hs.514941
MOV10
1p13.2





leukemia virus 10,





homolog (mouse)


0.0001583
2.899
232406_at
Jagged 1 (Alagille
Hs.224012
JAG1
20p12.1-p11.23





syndrome)


0.0001589
2.919
1556138_a_at
Collagen, type V, alpha 1
Hs.210283
COL5A1
9q34.2-q34.3


0.0001597
0.435
200651_at
guanine nucleotide
Hs.5662
GNB2L1
5q35.3





binding protein (G





protein), beta polypeptide





2-like 1 (GNB2L1),





mRNA.


0.0001618
2.498
241809_at
Hypothetical protein
Hs.193406
LOC284465
1p13.2





LOC284465


0.0001619
0.456
201484_at
suppressor of Ty 4
Hs.439481
SUPT4H1
17q21-q23





homolog 1 (S. cerevisiae)





(SUPT4H1), mRNA.


0.0001621
0.526
225475_at
mesoderm induction early
Hs.21757
MIER1
1p31.2





response 1 homolog





(Xenopus laevis)





(MIER1), mRNA.


0.0001633
0.329
201529_s_at
replication protein A1,
Hs.461925
RPA1
17p13.3





70 kDa (RPA1), mRNA.


0.0001637
0.403
212199_at
Morf4 family associated
Hs.518608
MRFAP1L1
4p16.1





protein 1-like 1





(MRFAP1L1), transcript





variant 2, mRNA.


0.0001639
0.387
208796_s_at
cyclin G1 (CCNG1),
Hs.79101
CCNG1
5q32-q34





transcript variant 2,





mRNA.


0.0001644
3.72
238183_at
ESTs


0.0001644
3.205
228497_at
solute carrier family 22
Hs.125482
SLC22A15
1p13.1





(organic cation





transporter), member 15





(SLC22A15), mRNA.


0.0001645
2.614
204078_at
synaptonemal complex
Hs.446459
SC65
17q21.2





protein SC65 (SC65),





mRNA.


0.0001649
3.055
239367_at
brain-derived
Hs.502182
BDNF
11p13





neurotrophic factor





(BDNF), transcript





variant 6, mRNA.












0.0001678
2.961
227260_at
Transcribed locus
Hs.537755
 1













0.0001721
0.452
200074_s_at
ribosomal protein L14
Hs.446522
RPL14
3p22-p21.2





(RPL14), mRNA.


0.0001731
0.346
227529_s_at
A kinase (PRKA) anchor
Hs.371240
AKAP12
6q24-q25





protein (gravin) 12


0.0001747
0.451
229844_at
Transcribed locus
Hs.59368

 3


0.0001751
2.202
1568954_s_at
Unknown












0.0001753
2.848
1555243_x_at
Hypothetical gene
Hs.443072
8q21.2





supported by BC055092













0.0001794
0.326
218919_at
zinc finger, AN1-type
Hs.390395
ZFAND1
8q21.13





domain 1 (ZFAND1),





mRNA.


0.0001795
0.337
201674_s_at
A kinase (PRKA) anchor
Hs.463506
AKAP1
17q21-q23





protein 1 (AKAP1),





nuclear gene encoding





mitochondrial protein,





transcript variant 1,





mRNA.


0.0001802
2.365
202292_x_at
lysophospholipase II
Hs.533479
LYPLA2
1p36.12-p35.1





(LYPLA2), mRNA.


0.0001807
3.042
230850_at
Formin-like 3
Hs.179838
FMNL3
12q13.12


0.0001808
3.249
202016_at
mesoderm specific
Hs.270978
MEST
7q32





transcript homolog





(mouse) (MEST),





transcript variant 3,





mRNA.


0.0001816
0.269
209305_s_at
growth arrest and DNA-
Hs.110571
GADD45B
19p13.3





damage-inducible, beta





(GADD45B), mRNA.


0.0001825
2.505
238714_at
RAB12, member RAS
Hs.270074

18p11.22





oncogene family


0.0001828
0.441
225352_at
translocation protein 1
Hs.529591
TLOC1
3q26.2





(TLOC1), mRNA.












0.0001833
0.509
235556_at
Transcribed locus,
Hs.445247
 5





weakly similar to





NP_703324.1 glutamic





acid-rich protein (garp)





[Plasmodium falciparum





3D7]


0.0001833
0.505
1555823_at
BS 3076
Hs.170421
14













0.000184
0.349
212188_at
potassium channel
Hs.109438
KCTD12
13q22.3





tetramerisation domain





containing 12 (KCTD12),





mRNA.


0.000184
2.716
212769_at
Transducin-like enhancer
Hs.287362
TLE3
15q22





of split 3 (E(sp1)





homolog, Drosophila)


0.0001862
2.329
219289_at
hypothetical protein
Hs.313917
FLJ20718
16q12.1





FLJ20718 (FLJ20718)





transcript variant 1,





mRNA.


0.0001871
2.234
229665_at
Hypothetical protein
Hs.44402
CSTF3
11p13





LOC283267










0.0001876
15.895
231597_x_at
ESTs, Weakly similar to T47135 hypothetical protein





DKFZp761L0812.1 [H. sapiens]












0.000189
2.944
1558426_x_at
Chromosome 7 open
Hs.534807
 7





reading frame 19













0.0001908
0.386
244050_at
similar to RIKEN
Hs.136247
LOC401494
9p21.3





4933428I03





(LOC401494), mRNA.


0.0001916
0.44
218311_at
mitogen-activated protein
Hs.468239
MAP4K3
2p22.1





kinase kinase kinase





kinase 3 (MAP4K3),





mRNA.


0.0001922
0.441
218373_at
fused toes homolog
Hs.380897
FTS
16q12.2





(mouse) (FTS), transcript





variant 2, mRNA.


0.0001939
0.429
203166_at
craniofacial development
Hs.461361
CFDP1
16q22.2-q22.3





protein 1 (CFDP1),





mRNA.










0.0001953
3.032
214110_s_at
ESTs, Highly similar to A43542 lymphocyte-specific protein 1





[H. sapiens]













0.0001962
2.652
229748_x_at
Hypothetical protein
Hs.487562
LOC285458
 4





LOC285458












0.0001971
3.714
240421_x_at
CDNA clone
Hs.547654
 4





IMAGE: 5268630













0.0001976
4.61
213905_x_at
Biglycan
Hs.821
BGN
Xq28


0.0001992
0.426
224812_at
3-hydroxyisobutyrate
Hs.406758
HIBADH
7p15.2





dehydrogenase





(HIBADH), mRNA.


0.0001997
0.46
200010_at
Ribosomal protein L11
Hs.388664
RPL11
1p36.1-p35


0.0002003
0.474
200022_at
ribosomal protein L18
Hs.515517
RPL18
19q13





(RPL18), mRNA.










0.0002005
4.122
216858_x_at














0.0002007
0.421
217773_s_at
NADH dehydrogenase
Hs.50098
NDUFA4
7p21.3





(ubiquinone) 1 alpha





subcomplex, 4, 9 kDa





(NDUFA4), nuclear gene





encoding mitochondrial





protein, mRNA.












0.0002018
2.22
1556835_s_at
Transcribed locus
Hs.548301
11













0.0002019
9.389
203936_s_at
matrix metallopeptidase 9
Hs.297413
MMP9
20q11.2-q13.1





(gelatinase B, 92 kDa





gelatinase, 92 kDa type IV





collagenase) (MMP9),





mRNA.


0.0002023
2.731
219279_at
dedicator of cytokinesis
Hs.46578
DOCK10
2q36.3





10 (DOCK10), mRNA.


0.0002042
0.49
230141_at
AT rich interactive
Hs.161000
ARID4A
14q23.1





domain 4A (RBP1-like)


0.0002053
0.39
204454_at
leucine zipper, down-
Hs.45231
LDOC1
Xq27





regulated in cancer 1





(LDOC1), mRNA.


0.0002057
0.112
206211_at
selectin E (endothelial
Hs.89546
SELE
1q22-q25





adhesion molecule 1)





(SELE), mRNA.


0.0002058
2.146
227214_at
Golgi associated PDZ
Hs.191539
GOPC
6q21





and coiled-coil motif





containing


0.000206
0.448
224754_at
Sp1 transcription factor
Hs.524461
SP1
12q13.1





(SP1), mRNA.












0.0002067
0.353
226873_at
Transcribed locus
Hs.548339
16













0.0002101
0.306
226688_at
chromosome 3 open
Hs.55131
C3orf23
3p21.33-p21.32





reading frame 23





(C3orf23), transcript





variant 1, mRNA.


0.0002108
0.459
222431_at
Spindlin
Hs.146804
SPIN
9q22.1-q22.3


0.0002111
0.507
226705_at
Fibroblast growth factor
Hs.264887
FGFR1
8p11.2-p11.1





receptor 1 (fms-related





tyrosine kinase 2, Pfeiffer





syndrome)


0.0002121
0.267
202350_s_at
matrilin 2 (MATN2),
Hs.189445
MATN2
8q22





transcript variant 2,





mRNA.


0.0002123
3.369
228331_at
Chromosome 11 open
Hs.502630
C11orf31
11q12.1





reading frame 31


0.0002151
2.308
226599_at
KIAA1727 protein
Hs.132629
KIAA1727
4q31.3





(KIAA1727), mRNA.


0.000216
0.472
229431_at
regulatory factor X-
Hs.24422
RFXAP
13q14





associated protein





(RFXAP), mRNA.


0.0002181
2.628
210365_at
Runt-related transcription
Hs.149261
RUNX1
21q22.3





factor 1 (acute myeloid





leukemia 1; aml1





oncogene)


0.0002196
3.303
238584_at
IQ motif containing with
Hs.129174
IQCA
2q37.2-q37.3





AAA domain


0.0002203
0.512
201960_s_at
MYC binding protein 2
Hs.151411
MYCBP2
13q22





(MYCBP2), mRNA.


0.0002206
2.459
236715_x_at
uveal autoantigen with
Hs.108049
UACA
15q22-q24





coiled-coil domains and





ankyrin repeats (UACA),





transcript variant 1,





mRNA.


0.0002226
3.297
213979_s_at
C-terminal binding
Hs.208597
CTBP1
4p16





protein 1 (CTBP1),





transcript variant 1,





mRNA.


0.0002231
0.271
208703_s_at
amyloid beta (A4)
Hs.370247
APLP2
11q24





precursor-like protein 2





(APLP2), mRNA.


0.0002234
0.507
202536_at
chromatin modifying
Hs.476930
CHMP2B
3p12.1





protein 2B (CHMP2B),





mRNA.


0.0002241
4.333
214715_x_at
zinc finger protein 160
Hs.467236
ZNF160
19q13.41





(ZNF160), transcript





variant 1, mRNA.


0.0002246
0.401
202364_at
MAX interactor 1
Hs.501023
MXI1
10q24-q25





(MXI1), transcript variant





3, mRNA.


0.0002266
2.374
221943_x_at
ribosomal protein L38
Hs.380953
RPL38
17q23-q25





(RPL38), mRNA.


0.0002277
0.153
1552767_a_at
heparan sulfate 6-O-
Hs.385956
HS6ST2
Xq26.2





sulfotransferase 2





(HS6ST2), mRNA.










0.0002281
3.22
241223_x_at
ESTs, Weakly similar to ALU1_HUMAN ALU SUBFAMILY J





SEQUENCE CONTAMINATION WARNING ENTRY [H. sapiens]













0.0002292
0.399
207132_x_at
prefoldin 5 (PFDN5),
Hs.288856
PFDN5
12q12





transcript variant 1,





mRNA.


0.0002299
2.574
218739_at
abhydrolase domain
Hs.19385
ABHD5
3p21





containing 5 (ABHD5),





mRNA.


0.0002299
3.3
217497_at
Endothelial cell growth
Hs.546251
ECGF1
22q13





factor 1 (platelet-derived)


0.0002316
2.781
218193_s_at
golgi transport 1 homolog
Hs.62275
GOLT1B
12p12.1





B (S. cerevisiae)





(GOLT1B), mRNA.


0.0002317
0.315
209146_at
sterol-C4-methyl
Hs.105269
SC4MOL
4q32-q34





oxidase-like (SC4MOL),





transcript variant 2,





mRNA.


0.0002325
3.134
201487_at
cathepsin C (CTSC),
Hs.128065
CTSC
11q14.1-q14.3





transcript variant 1,





mRNA.


0.0002331
3.007
202028_s_at
ribosomal protein L38
Hs.380953
RPL38
17q23-q25





(RPL38), mRNA.


0.0002336
3.872
217715_x_at
ESTs


0.0002337
2.486
1553570_x_at
Unknown












0.0002364
0.558
201178_at
F-box protein 7 (FBXO7), transcript
FBXO7
22q12-q13





variant 2, mRNA.













0.0002365
6.732
210809_s_at
periostin, osteoblast
Hs.136348
POSTN
13q13.3





specific factor (POSTN),





mRNA.


0.0002372
3.675
226997_at
CDNA FLJ10196 fis,
Hs.12680

 5





clone HEMBA1004776


0.0002389
0.316
226038_at
LON peptidase N-
Hs.180178
LONRF1
8p23.1





terminal domain and ring





finger 1 (LONRF1),





mRNA.


0.0002408
2.579
AFFX-BioDn-
Unknown




5_at


0.0002413
2.498
206857_s_at
FK506 binding protein
Hs.306834
FKBP1B
2p23.3





1B, 12.6 kDa (FKBP1B),





transcript variant 1,





mRNA.


0.0002425
0.201
229339_at
Myocardin
Hs.462257
MYOCD
17p11.2


0.0002453
3.977
242578_x_at
Solute carrier family 22
Hs.242721
SLC22A3
6q26-q27





(extraneuronal





monoamine transporter),





member 3












0.0002463
2.667
231882_at
CDNA FLJ10674 fis,
Hs.536634
22





clone NT2RP2006436


0.0002463
2.619
1556185_a_at
CDNA clone
Hs.287168
 7





IMAGE: 5260162













0.0002474
1.938
202573_at
casein kinase 1, gamma 2
Hs.181390
CSNK1G2
19p13.3





(CSNK1G2), mRNA.


0.0002491
0.533
57715_at
Family with sequence
Hs.241545
FAM26B
10pter-q26.12





similarity 26, member B












0.000252
4.351
239806_at
Transcribed locus
Hs.136017
 2













0.0002539
2.067
232145_at
hypothetical LOC388969
Hs.516159
LOC388969
2p11.2





(LOC388969), mRNA.












0.0002541
0.522
235570_at
CDNA FLJ36544 fis,
Hs.101689
 3





clone TRACH2006378













0.0002548
0.455
224605_at
HCV F-transactivated
Hs.173705
LOC401152
4q26





protein 1 (LOC401152),





mRNA.


0.000255
3.099
205463_s_at
Platelet-derived growth
Hs.376032
PDGFA
7p22





factor alpha polypeptide


0.0002565
0.51
209384_at
proline synthetase co-
Hs.304792
PROSC
8p11.2





transcribed homolog





(bacterial) (PROSC),





mRNA.










0.0002577
5.738
234753_x_at














0.0002578
0.348
235061_at
protein phosphatase 1K
Hs.291000
PPM1K
4q22.1





(PP2C domain





containing) (PPM1K),





mRNA.












0.0002619
0.279
235278_at
chromosome 20 open reading frame 133
C20orf133
20p12.1





(C20orf133), transcript variant 2,





mRNA.













0.0002624
0.147
218730_s_at
osteoglycin
Hs.109439
OGN
9q22





(osteoinductive factor,





mimecan) (OGN),





transcript variant 3,





mRNA.


0.000263
0.362
1554464_a_at
cartilage associated
Hs.517888
CRTAP
3p22.3





protein (CRTAP),





mRNA.


0.0002644
0.438
226994_at
DnaJ (Hsp40) homolog,
Hs.368078
DNAJA2
16q11.1-q11.2





subfamily A, member 2












0.000265
3.356
210679_x_at
B-cell CLL/lymphoma 7A
BCL7A
12q24.13













0.0002653
0.437
206621_s_at
Williams-Beuren
Hs.520943
WBSCR1
7q11.23





syndrome chromosome





region 1 (WBSCR1),





transcript variant 2,





mRNA.


0.0002657
0.392
1558487_a_at
Transmembrane emp24
Hs.510745
TMED4
7p13





protein transport domain





containing 4


0.0002665
2.208
1568619_s_at
Hypothetical protein
Hs.530899
LOC162073
16p12.3





LOC162073


0.0002666
3.366
229795_at
Transcribed locus
Hs.48945

12


0.0002674
0.312
200906_s_at
palladin (KIAA0992),
Hs.151220
KIAA0992
4q32.3





mRNA.


0.0002675
2.764
202581_at
heat shock 70 kDa protein
Hs.274402
HSPA1B
6p21.3





1B (HSPA1B), mRNA.


0.0002697
0.479
225330_at
Insulin-like growth factor
Hs.20573
IGF1R
15q26.3





1 receptor


0.0002718
2.171
225480_at
chromosome 1 open
Hs.532749
C1orf122
1p34.3





reading frame 122





(C1orf122), mRNA.


0.0002728
0.412
227132_at
HSPC038 protein
Hs.374485
LOC51123
8q22.3





(LOC51123), mRNA.


0.0002731
0.533
200031_s_at
ribosomal protein S11
Hs.433529
RPS11
19q13.3





(RPS11), mRNA.












0.0002738
0.328
229994_at
MRNA; cDNA
Hs.379253
 1





DKFZp686J23256 (from





clone DKFZp686J23256)












0.0002746
3.124
207730_x_at
hypothetical protein FLJ20700
FLJ20700
19p13.3













0.0002751
2.866
235327_x_at
UBX domain containing
Hs.516018
UBXD4
2p23.3





4 (UBXD4), mRNA.


0.0002768
5.302
212236_x at
keratin 17 (KRT17),
Hs.2785
KRT17
17q12-q21





mRNA.


0.0002777
1.816
218159_at
chromosome 20 open
Hs.471975
C20orf116
20p13





reading frame 116





(C20orf116), mRNA.


0.0002779
0.309
202119_s_at
copine III (CPNE3),
Hs.191219
CPNE3
8q21.3





mRNA.


0.000278
2.474
225636_at
signal transducer and
Hs.530595
STAT2
12q13.3





activator of transcription





2, 113 kDa (STAT2),





mRNA.












0.0002844
3.057
224667_x_at
Transcribed locus
Hs.558150














0.0002846
2.854
233406_at
KIAA0256 gene product
Hs.9997
KIAA0256
15q21.1


0.0002862
0.464
225941_at
Eukaryotic translation
Hs.476782
EIF4E3
3p14





initiation factor 4E





member 3


0.0002867
0.43
212368_at
PREDICTED: zinc finger
Hs.485892
ZNF292
 6





protein 292 (ZNF292),





mRNA.









Example 5
Array Validation
This example provides further support for the use of the endothelial cell tumor-associated molecules provided in Examples 3 and 4 to identify ovarian tumor endothelial cells.
To substantiate the findings provided by the microarray analysis described in Examples 3 and 4, a series of 17 genes were selected at random spanning a range of fold-changes (3.6 to 155.3; FIG. 2). Of 17 primer sets, 15 yielded specific qRT-PCR products when analyzed using Universal Human Reference RNA (Stratagene, La Jolla, Calif.), with 13 reaching statistical significance in tumor (n=10) and normal (n=5) isolates (p<0.05) including PLXDC1, ARBB2, HES4, PGF, EGFL6, ADAM12, COL5A3, COL18A1, PCOLCE, PMAIP1, CENTA2, TMEPAI, and NPTX2. In order to substantiate the pathway analysis (presented below), a second set of genes implicated in endothelial tumor cell signaling was assessed. From a series of 12 genes, suitable primer sets were obtained for 10 genes. All 10 pathway members were successfully validated (p<0.05) including FYN, VAV2, ECGF1, PTK2, TNFAIP6, EZH2, STC1, MMP9, JAG1, and CSPG2 (FIG. 1).
To further examine whether the gene expression alterations identified by the microarray analysis also occur at the protein level, immunohistochemical staining was performed for selected proteins on 5 normal ovaries and 5 invasive epithelial ovarian cancers. The microarray analysis identified FAK (PTK2; 3.1-fold), Fyn (4.7-fold), MMP-9 (9.4-fold), β2-arrestin (4.8-fold), Jagged1 (4.3-fold), and PLXDC1 (10.2-fold) as being significantly increased in tumor-associated endothelial cells, and these changes were validated by real-time RT-PCR.
Immunohistochemical-peroxidase staining confirmed that both FAK and Fyn were indeed overexpressed in the tumor-associated endothelial cells in all samples. There were no obvious differences in protein expression between arterioles and venules. Similarly, increased expression of MMP-9, β2-arrestin, Jagged1, and PLXDC1 was also confirmed at the protein level (FIG. 2). These results provide further support for the use of the specific endothelial cell tumor-associated molecules provided in Examples 3 and 4 to identify ovarian tumor endothelial cells.
Example 6
Modulation of Endothelial Cell Tumor-Associated Molecules
This example illustrates signaling pathways that are modulated in tumor endothelium and their functional significance.
Ovarian epithelial carcinomas arise from molecular events occurring in the epithelial layer, which affect changes in gene expression within surrounding non-epithelial cell populations. For endothelial cells, this altered signaling environment stimulates proliferation, migration, and tumor vascularization. To identify epithelial genes that may be responsible for these changes and the endothelial signaling pathways that are impacted, a series of laser microdissected papillary serous epithelial cell isolates and ovarian surface epithelial brushings were compared, as previously described (Bonome et al., Cancer Res. 65: 10602-10612, 2005). Pathway diagrams were generated using Pathway Assist version 3.0 software. The genes comprising the pathway indicate involvement in endothelial cell proliferation, tube-formation, and cell motility.
To test the biological significance of some of these genes, three genes were selected—EZH2, Jagged1, and PTK2 EZH2 plays an important role in many biological processes and is downstream of Akt activation, making it a potential anti-angiogenic target. siRNA was used to inhibit EZH2 expression (FIG. 3A) in HUVEC cells and its effects on tube formation (FIG. 3D) and migration (FIG. 3E) were examined. In comparison to control non-silencing siRNA, EZH2 silencing resulted in an 85% decrease in endothelial tube-formation on Matrigel (FIG. 3D) EZH2-targeted siRNA completely blocked VEGF-stimulated migration of HUVEC cells (FIG. 3E). Similarly, to determine the functional relevance of Jagged1 for endothelial cell function, the effects of inhibiting Jagged1 expression with siRNA were evaluated (FIG. 3B) on tube-formation (FIG. 3D) and migration (FIG. 3F). Jagged1-targeted siRNA reduced tube-formation by 80% (FIG. 3D) and blocked VEGF-stimulated HUVEC migration (FIG. 3F). Similar results were noted with PTK2 expression inhibition with PTK2-targeted siRNA (FIGS. 3D and 3G). These data indicate that the novel differentially expressed genes in the tumor-associated endothelial cells play functionally significant roles in angiogenesis.
The ability of siRNA to be delivered directly into ovarian tumor cells was investigated by staining tumor tissues with (A) primary rat anti-mouse CD31 antibody to detect endothelial cells and (B) anti-f4/80 to detect scavenging macrophages and then Alexa 488-tagged secondary antibody. Fluorescent siRNA was not only trapped onto blood vessels, but was also effectively delivered deep into tumor parenchyma. Macrophages were observed to surround nests of tumor cells that contained perinuclear siRNA, and had less amount of siRNA compared to tumor cells suggesting that siRNAs were delivered directly into the tumor cells.
The effect of mouse EZH2 siRNA on EZH2-expression in mouse ovarian endothelial cells was also determined. Cells were trypsinized at different time intervals (24 h, 48 h and 72 h) after transfection with mouse EZH2 siRNA and checked for EZH2 mRNA down regulation using RT-PCR analysis. As illustrated in FIG. 4, EZH2 gene expression was significantly decreased after 24 hours of treatment, indicating that the administered siRNA was capable of down-regulating EZH2 mRNA in vitro.
Example 7
Inhibition of Tumor Growth and Vascularization in a Mouse Model
This example describes methods for significantly reducing ovarian tumor growth and vascularization in a mouse model. One of skill in the art will appreciate that similar methods can be used in other mammals and other siRNAs can be used in place of those described herein. Further, a conversion formula known to those of skill in the art can be employed to determine the appropriate doses in other mammals, including humans.
Nude mice were injected (via i.p.) with either 2.5×105 HeyA8 or 1.0×106 SKOV3ip1 cells. Mice were randomly divided into 4 groups: 1) control siRNA-chitosan, 2) mouse EZH2 siRNA-chitosan, 3) Human EZH2 siRNA-chitosan, and 4) combination of mouse plus human EZH2 siRNA-chitosan. Therapy was started on the seventh day by injecting chitosan siRNA twice weekly (150 μg/kg). Therapy was started on the seventh day by injecting chitosan siRNA twice weekly (150 μg/kg). Animals were sacrificed when mice became moribund (3-5 weeks after cell injection). Mouse weight, tumor weight, number of tumor nodules and tumor location were recorded. As illustrated in FIGS. 5 and 6, mice treated with mouse-EZH2 siRNA exhibited significant decrease in tumor burden compared to control siRNA (70% and 42% reduction in tumor weight of HeyA8 and SKOV3ip1 respectively, p=0.05). Human EZH2 siRNA also reduced the tumor burden (50% in HeyA8 and 24% reduction in SKOV3ip1, p=0.05 of only HeyA8 tumors) compared to mouse targeted EZH2 siRNA. However, the greatest reduction was observed when treating the mouse with a combination of mouse plus human EZH2 siRNA (84% and 65% reduction in HeyA8 and SKOV3ip1 tumors, respectively, (p=0.001). In the case of tumor nodules, again combination of mouse plus human EZH2-siRNA group consistently produced fewer tumor nodules with 75% and 53% reduction compared to control group (p=0.05). The effect of EZH2 siRNA on microvessel density was determined by harvesting tumors from the four different groups stated above and staining such tumors for CD31. The mouse targeted EZH2 siRNA group showed decreased number of blood vessels compared to human EZH2 siRNA and control siRNA treated tumors. Microvessel density of the combination treated group using both mouse and human EZH2 siRNA was significantly reduced when compared to that of the control group. These studies demonstrate the ability of EZH2-targeted siRNA to inhibit tumor growth and vascularization in vivo.
Example 8
This example illustrates that increased EZH2 expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome and that the anti-angiogenesis effect of EZH2 silencing is mediated via silencing VASH1.
Material and Methods.
Human Ovarian Cancer Specimens.
One-hundred and thirty paraffin-embedded epithelial ovarian cancer specimens with available clinical outcome data and confirmed diagnosis by a board-certified gynecologic pathologist were obtained from the Karmanos Cancer Institute tumor bank. All patients were diagnosed from 1985 to 2004 following primary cytoreductive surgery. Slides of tumor samples were obtained for EZH2, CD34, and VEGF expression analysis. Clinical variables obtained for correlative analyses included age at diagnosis, tumor stage and grade, and vital status of patients relative to disease-specific survival at the time of chart review.

Cell Lines and Culture.

The HeyA8 and SKOV3ip1 human epithelial ovarian cancer cells were maintained as described previously. The derivation and characterization of the murine ovarian endothelial cells (MOEC) has been described previously. The EAhy926 endothelial hybridoma cell line was provided by Dr. Robert Danner, CCMD, NIH, and was maintained as described previously, with sodium hypoxanthine and thymidine (HT) supplement (Invitrogen, Carlsbad, Calif.) instead of sodium hypoxanthine aminopterin and thymidine (HAT) supplement (Invitrogen). HUVEC were purchased from Cambrex (Walkersville, Md.) and maintained with heparin and gentamicin/amphotericin-B, as previously described.

EZH2 Promoter Construct.

The EZH2 promoter was amplified by PCR from the Roswell Park Cancer Institute (RPCI) human BAC library 11, Clone-ID RP11-992C19 purchased from the Children's Hospital Oakland Research Institute (Oakland, Calif.), and then cloned into the pGL3-Basic Vector (Promega Corp., Madison, Wis.). The EZH2 promoter construct was amplified using primers (Table 6) with XhoI and HindIII restriction endonuclease sites added to the ends. Purified PCR product was then cloned upstream of the luc+ gene in the pGL3-Basic Vector (Promega Corp.) using XhoI and HindIII.








TABLE 6





Primers and siRNA sequences used.
















EZH2 promoter (Human):



5′-GATACTCGAGGTCGGGAGTTCGAGACCA-3′ (forward; SEQ ID NO: 6)


5′-GTTTAAGCTTACTCGCGTTGTTCCCGCG-3′ (reverse; SEQ ID NO: 7)


 


VASH1 promoter (Human):


5′-CATGGGAGGGCTTGATGAAGG-3′ (forward; SEQ ID NO: 8)


5′-GCCTAGTCCATGCTGACCTTG-3′ (reverse; SEQ ID NO: 9)


 


Real time quantitative RT-PCR and ChIP assay:


Murine EZH2:


5′-GCTGAGCGTATAAAGACACC-3′ (forward; SEQ ID NO: 10)


5′-TCTACATCCTCAGTGGGAAC-3′ (reverse; SEQ ID NO: 11)


 


Human EZH2:


5′-TCATGCAACACCCAACAC-3′ (forward; SEQ ID NO: 12)


5′-CACAACCGGTGTTTCCTC-3′ (reverse; SEQ ID NO: 13)


 


Murine VASH1:


5′-CATCAGGGAGCTGCAGTACA-3′ (forward; SEQ ID NO: 14)


5′-CCCAGCTTCACCTTCTTCAG-3′ (reverse; SEQ ID NO: 15)


 


Human VASH1:


5-CATGGGAGGGCTTGATGAAGG-3′ (forward; SEQ ID NO: 16)


5′-CAAGGTCAGCATGGACTAGGC-3′ (reverse; SEQ ID NO: 17)


 


Murine E2F1:


5′-TGGATCTGGAGACTGACCAT-3′ (forward; SEQ ID NO: 18)


5′-AGTTGCAGCTGTGTGGTACA-3′ (reverse; SEQ ID NO: 19)


 


Murine E2F2:


5′-GCTCCTGACCAAGAAGTTCA-3′ (forward; SEQ ID NO: 20)


5′-GCAATCACTGTCTGCTCCTT-3′ (reverse; SEQ ID NO: 21)


 


Murine E2F3:


5′-TGCAGTCTGTCTGAGGATGG-3′ (forward; SEQ ID NO: 22)


5′-GAGGCCAGAGGAGAGAGGTT-3′ (reverse; SEQ ID NO: 23)


 


Murine E2F4:


5′-AAGAACTGGACCAGCACAAG-3′ (forward; SEQ ID NO: 24)


5′-ACTATCCAGCAGTGCAGAGG-3′ (reverse; SEQ ID NO: 25)


 


Murine E2F5:


5′-AGTTGTGGCTACAGCAAAGC-3′ (forward; SEQ ID NO: 26)


5′-GGAGAAAGCCGTAAAAGAGG-3′ (reverse; SEQ ID NO: 27)


 


SiRNA target sequences:


Nonsilencing control siRNA:


5′-TTCTCCGAACGTGTCACGT[dT] [dT]-3′ (Sense; SEQ ID NO: 28)


5′-ACGTGACACGTTCGGAGAA [dT] [dT]-3′ (Antisense; SEQ ID NO: 29)


 


Human EZH2 siRNA: Validated sequence


Murine EZH2 siRNA1:


5′-GCTCTTACTGCTGAGCGTA[dT] [dT]-3′ (Sense; SEQ ID NO: 30)


5′-TACGCTCAGCAGTAAGAGC [dT] [dT]-3′ (Antisense; SEQ ID NO: 31)


 


Murine EZH2 siRNA2:


5′-GAGCAAAGCTTGCATTCAT[dT] [dT]-3′ (Sense; SEQ ID NO: 32)


5′-ATGAATGCAAGCTTTGCTC [dT] [dT]-3′ (Antisense; SEQ ID NO: 33)


 


Murine EZH2 siRNA3:


5′-CATTGGTACTTACTACGAT[dT] [dT]-3′ (Sense; SEQ ID NO: 34)


5′-ATCGTAGTAAGTACCAATG [dT] [dT]-3′ (Antisense; SEQ ID NO: 35)


 


Human VASH1 siRNA:


5′-GCGATGACTTCCGCAAGGA[dT] [dT]-3′ (Sense; SEQ ID NO: 36)


5′-TCCTTGCGGAAGTCATCGC [dT] [dT]-3′ (Antisense; SEQ ID NO: 37)


 


Murine VASH1 siRNA:


5′-GTGAGCTCGTGCTGGACTA[dT] [dT]-3′ (Sense; SEQ ID NO: 38)


5′-TAGTCCAGCACGAGCTCAC [dT] [dT]-3′ (Antisense; SEQ ID NO: 39)


 


Murine E2F3 siRNA:


5′-GTTTCTACAGCAAACCTCT[dT] [dT]-3′ (Sense; SEQ ID NO: 40)


5′-AGAGGTTTGCTGTAGAAAC [dT] [dT]-3′ (Antisense; SEQ ID NO: 41)


 


Murine E2F5 siRNA:


5′-CAATTGCTTTCATGGTGAT[dT] [dT]-3′ (Sense; SEQ ID NO: 42)


5′-ATCACCATGAAAGCAATTG [dT] [dT]-3′ (Antisense; SEQ ID NO: 43)









Luciferase Reporter Assay.

Relative activity of the EZH2 promoter in the EAhy926 cell line was determined by luciferase reporter assay. Cells were transfected in low-serum medium (0.5% serum) with the firefly luciferase plasmid, either empty vector (pGL3-Basic) or the EZH2 promoter construct vector (EZH2prom-pGL3-Basic), in 12-well plates using Effectene® Transfection Reagent from Qiagen (Valencia, Calif.). The primer sequence of EZH2 promoter are given in Table 6. Cells were then maintained in low-serum medium for 18 hours, washed in warm 1× phosphate-buffered saline (PBS), and treated in triplicate at 37° C. for 6 hours. Treatments included recombinant human (rh) EGF (EGF; 25 ng/mL; Invitrogen) and rhVEGF165 (VEGF; 50 ng/mL; Peprotech, Rocky Hill, N.J.), each in fresh medium plus 0.5% serum, fresh complete medium plus 10% serum, and conditioned media from immortalized ovarian surface epithelium (IOSE120) and from papillary serous ovarian cancer cell lines (OVCA420 and SKOV3). Medium in control wells (pGL3-Basic transfectants) was not changed on the day of treatment. Following treatment, cells were washed briefly in cold 1×PBS and lysates were collected and processed using the Dual-Luciferase® Reporter Assay System (Promega Corp.). Firefly luciferase readings were averaged and normalized to pGL3-Basic control readings for percent fold changes.

Chromatin Immunoprecipitation (ChIP) Assay.

HUVEC were cultured in low serum medium (0.5% serum) for 18 h and then treated with or without VEGF (50 ng/mL) for 6 hours. After treatment, ChIP assays were performed using EZ ChIPTM kit (Milllipore, Temecula, Calif.) as described by the manufacturer. Briefly, cross-linked cells were collected, lysed, sonicated and subsequently subjected to immunoprecipitation with EZH2 (Cell signaling) antibody or mouse IgG (mIgG) control. Immunocomplexes were collected with protein G agarose beads and eluted. Cross-links were reversed by incubating at 65° C. DNA then was extracted and purified for PCR using primers (see Table 6) corresponding to the 3800 to 3584 base pairs upstream of the VASH1 transcription start site.

Real Time Quantitative RT-PCR.

Relative expression of EZH2 and VASH1 mRNA in HUVEC and MOEC cells was determined by real-time quantitative RT-PCR. Cells were seeded at 1.0×104 cells per well in 96-well plates in complete medium and incubated at 37° C. for 24 hours, and then in low-serum medium (0.5% serum) for 18 hours, minus EGF and VEGF supplements where appropriate. After washing with warm PBS, cells were treated in triplicate at 37° C. for 6 hours with EGF (25 ng/mL) and VEGF (50 ng/mL), each in fresh medium (lacking supplemental EGF or VEGF) with no serum, fresh complete medium plus 2% serum, and conditioned media. Relative expression of VASH1 mRNA in MOEC cells was determined by transfecting cells with EZH2 mouse siRNA. Samples were collected after 72 hours of transfection. Expression of E2F transcription factors and levels of EZH2 in E2F transcription factors silenced endothelial cells (MOEC) was determined using specific siRNA for E2F transcriptional factors. Real-time quantitative RT-PCR was performed using 50 ng total RNA isolated from treated cells using the RNeasy Mini Kit (Qiagen). (SiRNA and primer sequences are given in Table 6). Relative expression values were obtained using the average of three reference genes and the 2−ΔΔCT method as described previously, and normalized to control for percent fold changes.

SiRNA Constructs and Delivery.

SiRNA nonsilencing control or EZH2 Hs siRNA were purchased from Qiagen and EZH2 Mm siRNA from Dharmacon (Chicago, Ill.). A nonsilencing siRNA that did not share sequence homology with any known human mRNA based on a BLAST search was used as control for target siRNA, and the same sequence with Alexa-555 tag was used to determine the uptake and distribution in tumor and various organs when given in vivo. In vitro transient transfection was performed as described previously and cells were harvested to measure EZH2 protein downregulation by Western blot analysis. (SiRNA sequences are given in Table 6).

DNA Extraction and Methylation Analysis.

DNA was extracted from the EZH2 silencing cells and mock cells using standard phenol-chloroform methods. Methylation analysis was done using a methylation kit (EZ-96 gold; Zymo Research, Orange, Calif.). MethPrimer software was used for the prediction of CpG island of Mm VASH1 (ACCESSION AB284948; VERSION AB284948.1; GI: 118442795) and design of methylation specific primers. The sequence of primers for methylated VASH1 at promoter region was TTAGGGATTTACGTATCGACGT (forward; SEQ ID NO: 44); AAACGACAAACTCCAACCG (reverse; SEQ ID NO: 45); and for unmethylated VASH1 promoter was TTTTTTTTAGGGATTTATGTATTGATGT (forward; SEQ ID NO: 46); CTAAACAACAAACTCCAACCACA (reverse; SEQ ID NO: 47). The PCR conditions were 94° C. for 5 min with hot start, then 94° C. for 45 second, 56° C. for 45 second, and 72° C. for 45 second, repeated for 40 cycles. Image analysis (Scion Image for Windows) was used for semi-quantitative measurement of methylated and unmethylated VASH1. Methylated VASH1 was normalized by unmethylated VASH1. The studies were repeated 3 times.

Cell Proliferation, Migration and Tube Formation Assay:

Cells were seeded in 96-well plates at 1×103 cells/well in replicates of 12. After 48 hours, cell growth was arrested; 36 hours after growth arrest, the specific mediators were added to untreated cells. Proliferation is assessed by the MTT dye technique, as previously described. The Membrane Invasion Culture System (MICS) chamber was used to measure the in vitro migration ability of cells.

Orthotopic In Vivo Model of Ovarian Cancer and Tissue Processing.

Female athymic nude mice (NCr-nu) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, Md.) and maintained as previously described. Tissue specimens were fixed either with formalin or OCT (optimum cutting temperature; Miles, Inc., Elkhart, Ind.) or were snap frozen.

To assess tumor growth for long-term therapy experiments, treatment began 1 week after intraperitoneal injection of tumor cells. Mice were divided into 4 groups (n=10 mice per group): (a) control siRNA/CH, (b) EZH2 Hs siRNA/CH (c) EZH2 Mm siRNA/CH, and (d) EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH. VASH1 gene silencing effects was determined using same cells and mice were divided into 6 groups. (a) Control siRNA/CH, (b) EZH2 Mm siRNA1/CH (c) EZH2 Mm siRNA2/CH, (d) EZH2 Mm siRNA3/CH (e) VASH1 Mm siRNA/CH and (f) VASH1 Mm siRNA/CH plus EZH2 Mm siRNA/CH. Each siRNA was given twice weekly at a dose of 150 μg/kg body weight. Treatment continued until mice became moribund (typically 4 to 5 weeks following tumor-cell injection) in any group. At the time of sacrifice, mouse weight, tumor weight, number of nodules, and distribution of tumors were recorded. The individuals who performed the necropsies, tumor collections, and tissue processing were blinded to the treatment group assignments.
Immunofluorescence and Confocal Microscopy.
Localization of EZH2 and CD31 was performed using frozen tissue. Tumors collected after 48 hours of single injection of control siRNA/CH, or EZH2 Hs siRNA/CH, or EZH2 Mm siRNA/CH, or EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH and stained for CD31 and EZH2. Staining for CD31 and desmin was done as described previously. Pericyte coverage was determined by the percent of vessels with 50% or more coverage by the green fluorescence of associated desmin-positive cells in 5 random fields at ×200 magnification for each tumor.

Western Blot Analysis.

Western blot analysis for EZH2 expression, histone3 (Lys27) methylation in vitro and EZH2 expression for in vivo samples was performed as previously reported. Tumors were collected at various time points (after 24, 48, 72 and 96 hours of single injection of control siRNA/CH, or EZH2 Hs siRNA/CH, or EZH2 Mm siRNA/CH, or EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH) and lysed to analyze protein levels using Western blotting.

EZH2 Gene Silencing in MOEC.

Relative expression of EZH2 mRNA in MOEC was determined by transfecting cells with control or EZH2 Mm siRNA and harvested after 72 hours of transfection. Real-time quantitative RT-PCR was performed using 50 ng total RNA isolated from treated cells using the RNeasy Mini Kit (Qiagen). Primer sequences are given in the Table 7. Relative expression values were obtained using the average of 3 reference genes and the 2−ΔΔCT method as described previously, and normalized to control for percent fold changes.








TABLE 7







Characteristics of tumors after treatment


with EZH2 siRNA/CH and VASH1 siRNA/CH.












Median no.





nodules
p -value


Cell line
Treatment
(range)
(vs. control)














HeyA8
Control siRNA/CH
19.0
(9-25)




EZH2 Mm siRNA1/CH
2.0
(0-8)
0.01* 



EZH2 Mm siRNA2/CH
9.0
(0-11)
0.046*



EZH2 Mm siRNA3/CH
9.0
(2-19)
ns



VASH1 Mm siRNA/CH
16.0
(12-21)
0.01**



EZH2 Mm siRNA1/CH plus
20.0
(7-32)
0.02**



VASH1 Mm siRNA/CH





*vs control siRNA/CH;


**vs EZH2siRNA/CH






Immunohistochemical Staining.

Detection of microvessel density was performed using formalin-fixed, paraffin-embedded tumor sections (8 μm thickness) as previously described. To quantify MVD, the number of blood vessels staining positive for CD31 was recorded in 10 random 0.159 mm2 fields at ×200 magnification. All staining was quantified by 2 investigators in a blinded fashion. Immunohistochemistry for EZH2 (1:400 dilution, Zymed, San Francisco, Calif.), CD34 (1:20 dilution, BioGenex Laboratories, San Ramon, Calif.), VEGF (1:100 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) was performed, as described previously. A combined score that was based on the staining intensity and the percentage of cells stained was used to assign a final score.

Statistical Analysis.

Differences in continuous variables such as mean body weight, tumor weight, and proliferation (PCNA) were analyzed using the Mann-Whitney rank sum test. Statistical analyses were performed using SPSS 12.0 for Windows® (SPSS Inc., Chicago, Ill.). A 2-tailed p<0.05 was considered statistically significant. Kaplan-Meier survival plots were generated and comparisons between survival curves were made using the log-rank statistic.

Conditioned Media.

Conditioned media were obtained as follows: IOSE120, OVCA420 and SKOV3 cells were grown in 100 mm culture dishes at 37° C. until 80% confluent. Cells were then washed briefly in warm 1×PBS. Then, 5 mL of low-serum, complete HUVEC cell medium (0.5% serum) was added to the dishes and the cells were incubated at 37° C. for 16 hours. Supernatants (conditioned media) were then collected in a syringe and passed through a 0.45 micron filter and stored at −80° C. until needed.

Preparation of siRNA-Incorporated Ch (siRNA/Ch) Nanoparticles.

CH (Molecular weight 50-190 kDa), sodium tripolyphosphate (TPP), and agarose were purchased from Sigma Co. (St. Louis, Mo.). SiRNA/CH nanoparticles were prepared based on ionic gelation of anionic TPP and siRNA with cationic CH. The formulation of the siRNA/CH nanoparticles is shown in FIG. 11A. Briefly, various concentrations of CH solution was obtained by dissolving CH in 0.25% acetic acid and nanoparticles were spontaneously generated by the addition of TPP (0.25% w/v) and siRNA (1 μg/μL) to CH solution under constant stirring at room temperature. After incubating at 4° C. for 40 min, siRNA/CH nanoparticles were collected by centrifugation (Thermo Biofuge, Germany) at 12,000 rpm for 40 minutes at 4° C. The pellet was washed 3 times to remove unbound chemicals or siRNA and siRNA/CH nanoparticles were stored at 4° C. until used.

Characteristics of siRNA/CH Nanoparticles.

The size and zeta potential of the siRNA/CH nanoparticles were measured by light scattering with a particle size analyzer and Zeta Plus (size and zeta potential analyzer, Brookhaven Instrument Co., CA), respectively. To measure the loading efficiency of siRNA into CH nanoparticles, Alexa-555 fluorescent-labeled siRNA was incorporated into CH nanoparticles followed by centrifugation at 12,000 rpm for 40 minutes. The fluorescence intensity in the supernatant was measured at 590 nm using fluorescence spectrophotometer (Fluostar Optima, BMG Labtech Inc., Durham, N.C.). Additionally, the morphology of CH nanoparticles was confirmed by AFM.

Gel Retardation Assay.

The incorporation of siRNA into CH nanoparticles was determined by 4% agarose gel electrophoresis. Electrophoresis was carried out at a constant voltage of 100 V for 1 hour in 0.5% TAE buffer containing 0.5 μg/mL ethidium bromide (EtBr). The siRNA bands were then visualized under a UV transilluminator (Fluor Chem 8900, Alpha Innotech, Madison, Wis.).

Stability Assay.

Stability of the siRNA-CH nanoparticles in 50% serum was characterized using 4% agarose gel electrophoresis. Either naked siRNA or siRNA/CH nanoparticles were mixed in a 1:1 ratio with fresh serum to get the 50% concentration and incubated at 37° C. Aliquots of 20 μL were collected at selected time intervals, loaded onto an agarose gel followed by electrophoresis to visualize intact siRNA.

Results

EZH2 Expression in Human Ovarian Carcinoma

The clinical significance of EZH2 was evaluated in 130 epithelial ovarian cancers. Increased tumoral EZH2 (EZH2-T) expression was noted in 66% of samples and increased expression in the vasculature (EZH2-Endo) was noted in 67% of the samples (FIG. 7A). Increased expression of EZH2-T and EZH2-Endo was significantly associated with high-stage (p values<0.001) and high-grade (p values<0.05; see Table 8) disease. Increased EZH2-T was significantly associated with decreased overall survival (median 2.5 years vs. 7.33 years, p values<0.001; FIG. 7B). Similarly, EZH2-Endo was predictive of poor overall survival (2.33 vs. 8.33 years, p<0.001; FIGS. 7C and 7D). On the basis of pathway-analysis predictions from the disclosed genomic profiling data comparing endothelial cells from epithelial ovarian cancer with those from normal ovarian tissues, the potential associations between EZH2 expression, VEGF expression and microvessel density (MVD) was examined. Increased VEGF expression was strongly associated with increased EZH2-Endo expression (p<0.001; FIGS. 7E and 7F). Moreover, increased EZH2-Endo expression was significantly associated with high MVD counts in the tumor (p<0.001; FIGS. 7G and 7H).








TABLE 8







Association of clinical and demographic features with


EZH2 in epithelial ovarian carcinoma.










EZH2-T
EZH2-Endo



overexpression
overexpression














No
Yes
p-value
No
Yes
p-value











Mean age 59.8 yrs (range 37-89 yrs)













Stage








Low (I/II)
20
9
<0.001
20
9
<0.001


High (III-IV)
41
108
<0.001
37
112
<0.001


Grade


Low
9
7
0.048
10
6
0.005


High
52
112
0.048
37
112
0.005


Histology


Serous
22
18
0.002
22
1
<0.001


Others
39
100
0.002
35
104
<0.001









VEGF Increases EZH2 Levels in Endothelial Cells

EAhy926 hybridoma endothelial cells were co-transfected with the Renilla luciferase plasmid and firefly luciferase plasmid either with or without the EZH2 promoter construct. Cells were then treated with VEGF, EGF, or conditioned media from ovarian cancer cell lines. EZH2 promoter activity was determined by the dual-luciferase assay. There was a significant increase in EZH2 promoter activity in endothelial cells in response to VEGF, EGF, and conditioned media (FIG. 8A). In order to examine changes in EZH2 message, HUVECs were treated as indicated above and expression of EZH2 mRNA was examined using quantitative real time RT-PCR. Control values were normalized using 3 housekeeping genes EZH2 mRNA expression levels were induced (by 130-240% fold change compared to control) in endothelial cells in response to VEGF, EGF, or the conditioned media (FIG. 8B). To examine the relationship between EZH2 and VEGF in human samples, the expression levels of both genes in 29 microdissected high-grade, serous papillary ovarian cancers were determined. Pearson's analysis showed a significant correlation between EZH2 and VEGF levels (p=0.03; FIG. 8C).
EZH2 Silencing Increases VASH1 in Endothelial Cells
To determine the mechanism by which EZH2 silencing could induce anti-angiogenic effects, a whole genome ChIP-on-ChIP analysis was performed. The findings indicate that an anti-angiogenic gene, vasohibin (VASH1) directly binds to EZH2. To validate this finding, a ChIP assay of EZH2 for the VASH1 promoter in endothelial cells in the presence or absence of VEGF was performed (FIG. 9A), which confirmed direct EZH2 binding to the VASH1 promoter. Next, we silenced the EZH2 gene in mouse ovarian endothelial cells (MOEC) using siRNA (FIG. 9B), which resulted in a 2.8 fold increase in VASH1 (FIG. 9C).
To determine the mechanism by which EZH2 regulates VASH1, methylation specific PCR was performed for detecting VASH1 methylation in endothelial cells in the presence of VEGF after silencing EZH2 VEGF treatment resulted in a 1.7 fold increase in VASH1 methylation compared to the controls. However, EZH2 silencing resulted in a 3.3 fold decrease in VASH1 methylation in the VEGF-treated MOEC cells (FIG. 9D). Specifically, EZH2 gene silencing by decreased histone 3 methylation at lysine 27 by 2.5 fold in endothelial cells (FIG. 9E).
E2F Mediated Regulation of EZH2 in Endothelial Cells.
The effect of VEGF on E2F1-5 in MOEC is provided in FIG. 10A. There was a significant increase in E2F1, E2F3 and E2F5 following treatment with VEGF (FIG. 10B). To determine which E2F transcription factors might be responsible for increasing EZH2 levels, the effects of VEGF after silencing either E2F1, 3 or 5 were determined EZH2 levels were significantly decreased in E2F3 and E2F5 silenced cells (FIG. 10B). To validate the binding of EZH2 promoter to E2F3 and E2F5 transcription factors, ChIP assays of EZH2 to these transcription factors were performed. E2F3 and E2F5 were bound to the EZH2 promoters, demonstrating that EZH2 is the direct target of the E2F transcription factors. The studies provide direct explanation for the anti-angiogenesis effects observed in response to EZH2 gene silencing.
VASH1 Gene Silencing Increases the Migration, Tube Formation In Vitro and the Tumor Growth In Vivo
To determine the role of VASH1 on angiogenesis, migration and tube formation studies were performed in MOEC and HUVEC by silencing the VASH1 gene in MOEC and HUVEC. MOEC cells were transfected with control and VASH1 siRNA for 48 hours and then resuspended in serum free media. 75,000 cells were plated on pre-gelatin and Matrigel coated Transwell inserts which were placed in the lower chamber of VEGF containing media. Migration and tube formation were significantly increased after VASH1 gene silencing (FIG. 10C); whereas no change in proliferation of cells.
Whether EZH2 silencing in vivo would affect tumor growth and angiogenesis was determined. Before conducting the EZH2 targeted in vivo experiments, CH nanoparticles for systemic delivery of siRNA into both tumor cells and tumor-associated vasculature were developed and characterize. Several formulations of CH with siRNA (siRNA/CH) were tested (FIG. 11A) and optimized (FIGS. 11B-11E; FIGS. 12A-12B; FIGS. 10A-10C) and the 3:1 ratio (CH:TPP) nanoparticles showed the greatest (75%) incorporation efficiency (FIG. 11B). Therefore, for all subsequent studies, siRNA/CH3 nanoparticles were used due to their small size, slight positive charge, and high incorporation efficiency of siRNA.
Prior to performing proof-of-concept in vivo efficacy studies, the efficiency of siRNA delivery into orthotopic ovarian tumors was tested. Non-silencing siRNA labeled with Alexa-555 was incorporated into CH nanoparticles and injected intravenously (i.v.) into mice bearing HeyA8 orthotopic tumors (17 days after intraperitoneal inoculation of tumor cells). Tumors were harvested at 15 hours and 3, 5 and 7 days (3 mice per time point) following injection and examined for extent of siRNA delivery. At all time points, punctated emissions of the siRNA were noted in the perinuclear regions of individual cells. SiRNA was noted in >80% of fields examined following a single intravenous injection. To confirm delivery of siRNA in the vasculature, slides were also stained for CD31. siRNA was delivered into the tumor-associated endothelial cells, suggesting potential applications for targeting the tumor vasculature. To confirm intracellular delivery of siRNA, 3-dimensional reconstructions of the tumors using confocal microscopy were created. Lateral views of the optical sections clearly demonstrated the presence of siRNA within the tumor cells (FIGS. 13A and 13B). However, very little siRNA was taken up by macrophages as determined by labeling tissues with f4/80. To examine the delivery of siRNA into other organs, sections of liver, lung, kidney, heart, spleen and brain were also examined, and siRNA delivery was detected in most of these organs.
To examine the in vivo effects of EZH2 gene silencing on tumor growth, EZH2 siRNA directed to either the human (tumor cells; EZH2 Hs siRNA/CH) or mouse (endothelial cells; EZH2 Mm siRNA/CH) sequence were utilized. The specificity of siRNA was confirmed by testing each siRNA in both mouse endothelial (MOEC) and human tumor (HeyA8) cells (FIG. 14). Following intravenous injection of either control siRNA/CH, EZH2 Hs siRNA/CH, EZH2 Mm siRNA/CH, or the combination of EZH2 targeted siRNAs into HeyA8 tumor-bearing mice (n=3 mice per group at each time point), tumors were harvested at different time points and examined for EZH2 protein levels EZH2 levels were decreased by 24 hours following single injection of EZH2 Hs siRNA/CH with return of expression to baseline expression levels after 96 hours (FIG. 13C). To determine the localization of EZH2 silencing following siRNA/CH administration, we performed dual immunofluorescence staining for EZH2 and CD31. This study further demonstrated that EZH2 Hs siRNA/CH resulted in EZH2 silencing in the tumor cells whereas EZH2 Mm siRNA/CH silenced EZH2 only in the tumor endothelial cells (FIG. 13D).
To determine the therapeutic efficacy of EZH2 gene silencing, a well-characterized orthotopic model of ovarian carcinoma was utilized. Seven days following injection tumor cells into the peritoneal cavity, mice were randomly allocated to 1 of 4 groups of 10 mice each: 1) control siRNA/CH, 2) EZH2 Hs siRNA/CH, 3) EZH2 Mm siRNA/CH and 4) combination of EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH. Mice were sacrificed when animals appeared moribund due to significant tumor burden (4 to 5 weeks after cell injection depending on the cell line).
Alexa-555 siRNA uptake into macrophages and to various organs was evaluated. Tumor tissues were collected after single injection of untagged control siRNA/CH or Alexa-555 siRNA/CH nanoparticles and stained with anti-f4/80 antibody to detect scavenging macrophages (green; middle or right panels). Macrophages were seen surrounding nests of tumor cells and had minimal siRNA uptake. Left panel demonstrates lack of natural autofluorescence following injection of untagged control siRNA/CH. Images were taken at original magnification ×200 (left and middle) and ×400 (right). Histological sections were made from the liver, kidney, lung, brain, and heart tissues that were collected after intravenous injection of 5 μg Alexa-555 siRNA/CH nanoparticles and exposed to hematoxylin and eosin (H&E) and Hoechst staining. Left panel represents H&E staining, middle panel represents natural auto-fluorescence of each tissue after a single injection of untagged control siRNA/CH and right panel denotes Alexa-555 siRNA/CH (red). All images were taken at original magnification ×200.
As shown in FIG. 13E and FIG. 15, treatment with EZH2 Mm siRNA/CH resulted in a significant decrease in tumor burden compared to control siRNA/CH (62% reduction in HeyA8; p<0.02 and 40% reduction in SKOV3ip1, p<0.03) EZH2 Hs siRNA/CH as a single-agent had modest effects on tumor growth (p<0.04 for HeyA8; and p<0.05 for SKOV3ip1) compared with control siRNA/CH. However, the greatest reduction was observed with the combination of EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH (83% reduction in HeyA8, p<0.001 and 65% reduction in SKOV3ip1, p<0.001). To test for potential off-target effects, we tested the efficacy of 3 additional mouse EZH2 siRNA sequences with similar effects on tumor growth.
To evaluate the effects of EZH2 on other parameters of tumor growth, we examined tumor incidence and number of nodules (Table 9 below). The combination of EZH2 Hs siRNA/CH plus EZH2 Mm siRNA/CH resulted in a significant reduction in tumor nodules in both HeyA8 (p=0.002 vs. control siRNA treated group) and SKOV3ip1 tumors (p=0.004 vs. control siRNA treated group). The decrease in tumor burden occurred despite having comparable tumor incidence. The mean mouse body weight was similar among the different groups, suggesting that feeding and drinking habits were not affected.







TABLE 9







Characteristics of tumors after treatment


with human and mouse EZH2 siRNA/CH












Median no.





nodules
p -value


Cell line
Treatment
(range)
(vs. control)














HeyA8
Control siRNA/CH
6.5
(3-11)




EZH2 Hs siRNA/CH
3.5
(1-11)
0.05



EZH2 Mm siRNA/CH
3.0
(1-9)
0.05



EZH2 Hs siRNA/CH plus
1.5
(1-7)
0.002



EZH2 Mm siRNA/CH


SKOV3iP1
Control siRNA/CH
16.0
(11-26)



EZH2 Hs siRNA/CH
16.0
(8-27)
ns



EZH2 Mm siRNA/CH
12.0
(1-17)
0.05



EZH2 Hs siRNA/CH plus
7.5
(2-27)
0.004



EZH2 Mm siRNA/CH









Effect of EZH2 Targeting on Tumor Vasculature and Proliferation

To determine the potential mechanisms underlying the efficacy of EZH2 silencing on ovarian tumors, its effects on several biological end points were examined, including MVD, pericyte coverage (desmin) and cell proliferation (PCNA). EZH2 Mm siRNA/CH and the combination therapy groups had significantly lower microvessel density (FIG. 16A) compared to the EZH2 Hs siRNA/CH and control siRNA/CH treated tumors. Pericyte coverage was increased in EZH2 Mm siRNA/CH and the combination groups compared to other 2 groups, suggesting greater vascular maturation (FIG. 16A). Combination treatment with EZH2 Hs siRNA/CH and EZH2 Mm siRNA/CH also resulted in a significant reduction in cell proliferation (FIG. 17).
To determine the requirement for VASH1 in mediating the anti-tumor effects of EZH2 silencing, the effects of VASH1 silencing in combination with EZH2 Mm siRNA/CH was determined. The anti-tumor effect of EZH2 silencing in the tumor vasculature was completely reversed by VASH1 silencing (FIG. 16B and see Table 7) suggesting that VASH1 is required for mediating the anti-tumor effects of EZH2 silencing.
Summary:
The present results provide a new understanding of the regulation of tumor angiogenesis. A novel mechanism by which VEGF increases EZH2 levels in the tumor vasculature was disclosed, which contributes to tumor angiogenesis by inactivating the anti-angiogenic factor, VASH1 via methylation of VASH1 gene and Histone 3 (Lys 27) (H3K27). Moreover, a novel and highly efficient method of gene silencing in the tumor cells as well as in the blood vessels that support their growth was developed and characterized. This approach was highly effective for EZH2 silencing in both compartments.
PcG proteins play a role in determining cell fate during both normal and pathologic processes. Two separate subsets of PcG complexes (PRC1 and PRC2) have been described in humans. PRC1 may be involved in maintenance of repression, whereas PRC2 plays a role in initiating repression. The PRC2 complex includes the EZH2, EED, and SUZ proteins. Altered expression of these proteins has been implicated in cancer pathogenesis. Increased EZH2 levels have been related to cancer cell proliferation and invasion. However, prior to the disclosed work, the role of EZH2 in angiogenesis was not known.
Angiogenesis is regulated by the balance of various pro-angiogenic stimulators, such as VEGF, and several angiogenesis inhibitors, such as angiostatin, endostatin, and antithrombin. On the basis of findings from genomic profiling of endothelial cells from ovarian cancer versus those from normal ovaries, it was discovered that EZH2 expression is significantly increased in tumor-associated endothelial cells. VEGF is well recognized as a pro-angiogenic factor in ovarian and other cancers. In the current study, it was shown for the first time that VEGF can directly increase EZH2 levels in endothelial cells, which in turn inactivating a potent anti-angiogenic factor, VASH1, via methylating VASH1 gene and H3K27. Silencing EZH2 gene resulted in demethylation of VASH1 gene and H3K27 in endothelial cells, which is consistent with other report indicating EZH2 directly controls DNA methylation of EZH2-targeted genes, concomitant with reducing H3K27. Therefore, through this study, a novel mechanism by which tumor angiogenesis is regulated was discovered and a rationale for pursuing EZH2 as a therapeutic target was provided.
While a number of attractive targets in tumor and endothelial cells have been identified, many of these are difficult to target with small molecule inhibitors and monoclonal antibodies. Therefore, RNA interference was employed as a means to target EZH2. Due to limited delivery of siRNA into the tumor-associated endothelial cells with this approach, additional nanoparticles were developed that would allow siRNA delivery into both tumor and tumor-associated endothelial cells. Chitosan (CH) is a naturally occurring polysaccharide with low immunogenicity and low toxicity. Here, CH was used because of its advantageous biological properties such as biodegradability, biocompatibility, and slight positive charge. These properties make use of CH for systemic in vivo siRNA delivery highly attractive. Indeed, the disclosed data demonstrate highly efficient delivery of siRNA incorporated into CH nanoparticles into both tumor and tumor-associated endothelial cells. Therefore, the present work provides an attractive method for systemic delivery of siRNA that could be developed for clinical applications.
Molecular and genetic manipulations have identified EZH2 as a key regulator of tumor angiogenesis here, but these effects do not rule out the possibility that EZH2 has oncogenic functions in the tumor cells. For example, EZH2 has been implicated in cellular transformation, proliferation, and avoidance of apoptosis. Such results imply that multiple signaling pathways likely convey the net effects of EZH2 in promoting tumor growth. However, to the extent that targeting tumor endothelial cells provides therapeutic benefit, interfering with EZH2 in the tumor and endothelial cells represents a novel strategy for treatment of ovarian and other cancers.
In summary, these studies illustrate that increased EZH2 expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation and indicates the presence of a paracrine circuit that promotes angiogenesis by methylating (histone H3; lysine 27) and silencing VASH1 EZH2 silencing in tumor cells and in the tumor-associated endothelial cells resulted in inhibition of angiogenesis and ovarian cancer growth. The anti-angiogenic effect was mediated by reactivating VASH1. Thus, these data support the potential for targeting EZH2 as a novel therapeutic approach
Example 9
Screening of Agents to Treat an Ovarian Tumor
This example describes methods that can be used to identify agents to treat an ovarian tumor.
According to the teachings herein, one or more agents for the use of treating an ovarian tumor, such as ovarian cancer can be identified by contacting an ovarian tumor endothelial cell with one or more test agents under conditions sufficient for the one or more test agents to alter the activity of at least one ovarian endothelial cell tumor-associated molecule listed in Tables 1, 2, 3, 4 or 5. The method also includes detecting the activity of the at least one ovarian endothelial cell tumor-associated molecule in the presence and absence of the one or more test agents. The activity of the at least one ovarian endothelial cell tumor-associated molecule in the presence of the one or more test agents is then compared to the activity in the absence of such agents to determine if there is differential expression of the at least one ovarian endothelial cell tumor associated molecule. Differential expression of the ovarian endothelial cell tumor-associated molecule indicates that the one or more test agents is of use to treat the ovarian tumor. For example, a test agent that reduces or inhibits the activity or expression of an ovarian endothelial tumor-associated molecule that is upregulated in ovarian tumor endothelial cells indicates that the test agent is of use to treat the ovarian tumor. Differential expression can be detected at the nucleic acid or protein level. An RNA expression product can be detected by a microarray or PCR by methods described above (see, for example, Example 1). A protein expression product can be detected by standard Western blot or immunoassay techniques that are known to one of skill in the art. However, the disclosure is not limited to particular methods of detection.
Example 10
Identification of Ovarian Endothelial Cell Tumor-Associated Molecule Inhibitors to Alter Tumor Growth and/or Vascularization
This example describes methods that can be used to identify ovarian endothelial cell tumor-associated molecule inhibitors that can be used to target specific genes involved in ovarian tumor growth and/or vascularization.
Based upon the teaching disclosed herein, iSynthetic siRNA molecules are generated against selected target genes, such as any of the ovarian endothelial cell tumor-associated up-regulated genes identified in Examples 2 through 5. In an example, the siRNA molecules are obtained from commercial sources. Knockdown efficiency of the siRNA molecules is assessed as indicated in Example 1. In an example, a significant knockdown efficiency is approximately 20%. As provided in Example 1, the effects of target gene siRNA's on tumor growth and vascularization can be determined by evaluating the effect of siRNA treatment on cell migration and tube formation in HUVECs.
In additional examples, cells are treated with two or more siRNAs (that target two or more genes). The IC50 values are compared (between target gene siRNA individually and in combination) to determine whether the knockdown effect on tumor growth and vascularization is cumulative or additive. siRNAs that reduce or decrease by approximately 20% the activity or expression of the targeted ovarian endothelial cell tumor-associated molecule which is upregulated in ovarian endothelial tumor cells are selected for further study.
Example 11
Effectiveness of an Ovarian Tumor Treatment
This example describes methods that can be used to identify effective ovarian tumor treatments.
Based upon the teachings disclosed herein, the effectiveness of an ovarian tumor treatment can be evaluated by determining the effectiveness of an agent for the treatment of an ovarian tumor in a subject with the ovarian tumor. In an example, the method includes detecting expression of an ovarian endothelial cell tumor-associated molecule in a sample from the subject following treatment with the agent. The expression of the ovarian endothelial cell tumor-associated molecule following treatment is compared to a control (a non-cancerous, ovarian endothelial cell). A reduction or inhibition of the expression or biological activity of the ovarian endothelial cell tumor-associated molecule which is upregulated in ovarian endothelial tumor cells following treatment indicates that the agent is effective for the treatment of an ovarian cancer in the subject. Alternatively, an increase in the expression or biological activity of an ovarian endothelial tumor-associated molecule that is downregulated in ovarian endothelial tumor cells following treatment indicates that the agent is effective for the treatment of the ovarian cancer in the subject. In a specific example, the method includes detecting and comparing the protein expression levels of the ovarian endothelial cell tumor-associated molecules. In other examples, the method includes detecting and comparing the mRNA expression levels of the ovarian endothelial cell tumor-associated molecules.
Example 12
Inhibition of Tumor Growth and/or Vascularization
This example describes methods that can be used to significantly reduce ovarian tumor growth, vascularization in a subject with ovarian cancer.
Based upon the teachings disclosed herein, an ovarian tumor, such as ovarian cancer can be treated by administering a therapeutically effective amount of a composition, wherein the composition comprises a specific binding agent that preferentially binds to one or more ovarian endothelial cell tumor-associated molecules provided in Tables 1 through 5, thereby inhibiting tumor growth and/or vascularization.
In an example, a subject who has been diagnosed with ovarian cancer is identified. In some examples, gene expression is screened to determine which genes are to be targeted. Following subject selection, a therapeutic effective dose of the composition including the specific binding agent is administered to the subject. For example, a therapeutic effective dose of a specific binding agent to one or more of the disclosed ovarian endothelial cell tumor-associated molecules is administered to the subject to reduce or inhibit tumor growth and/or vascularization. In an example, the specific binding agent is a siRNA. In another example, the specific binding agent is an antibody. In a further example, the specific binding agent is conjugated to a therapeutic agent such as a cytotoxin, chemotherapeutic reagent, radionucleotide or a combination thereof.
The amount of the composition administered to prevent, reduce, inhibit, and/or treat ovarian cancer or a condition associated with it depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g., ovarian cancer) in a subject without causing a substantial cytotoxic effect in the subject.
In one specific example, siRNAs are incorporated into the neutral liposome DOPC and injected intraperitoneal or intravenously at 150 μg/kg twice weekly for 2 to 3 weeks.
In another specific example, naked antibodies are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending upon the stage of the ovarian cancer. In an example, the antibodies are administered continuously. In another example, antibodies or antibody fragments conjugated to cytotoxic agents (immunotoxins) are administered at 50 μg per kg given twice a week for 2 to 3 weeks.
Example 13
Diagnosis of Metastatic Ovarian Cancer
This example describes particular methods that can be used to diagnose or prognose a metastatic ovarian tumor in a subject, such as metastatic ovarian cancer in a human. However, one skilled in the art will appreciate that similar methods can be used. In some examples, such diagnosis is performed before treating the subject (for example as described in Example 11).
Biological samples are obtained from the subject. If blood or a fraction thereof (such as serum) is used 1-100 μl of blood is collected. Serum can either be used directly or fractionated using filter cut-offs to remove high molecular weight proteins. If desired, the serum can be frozen and thawed before use. If a tissue biopsy sample is used, 1-100 μg of tissue is obtained, for example using a fine needle aspirate RNA or protein is isolated from the tissue using routine methods (for example using a commercial kit).
In one example, pro-angiogenic ovarian endothelial cell tumor-associated nucleic acid expression levels, such as nucleic acid expression levels of EZH2, are determined in a tumor sample obtained from the subject by microarray analysis or real-time quantitative PCR. In an example, the disclosed gene profile is utilized. In other examples, the amount of such molecules is determined at the protein level by methods known to those of ordinary skill in the art, such as Western blot or immunoassay techniques. The relative amount of pro-angiogenic ovarian endothelial cell tumor-associated molecules are compared to a reference value, such as a relative amount of such molecules present in a non-tumor sample from, wherein the presence of significantly greater amounts of pro-angiogenic ovarian endothelial cell tumor-associated molecules listed in Tables 1, 2, 4 and 5 (and indicated to be involved in angiogenesis) in the tumor sample as compared to the non-tumor sample (such as an increase of at least 2-fold, at least 3-fold, or at least 5-fold) indicates that the subject has a metastatic ovarian tumor, has an increased likelihood of an ovarian tumor metastasizing, has a poor prognosis, or combinations thereof. In other examples, a decrease in expression of those molecules listed in Table 3 (and involved in angiogenesis) indicates that the subject has a metastatic ovarian tumor, has an increased likelihood of an ovarian tumor metastasizing, has a poor prognosis, or combinations thereof. In some examples, relative amount of pro-angiogenic ovarian endothelial cell tumor-associated proteins and pro-angiogenic ovarian endothelial cell tumor-associated mRNA expression are determined in the same subject using the methods described above.
While this disclosure has been described with an emphasis upon particular embodiments, it will be obvious to those of ordinary skill in the art that variations of the particular embodiments may be used, and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Features, characteristics, compounds, or examples described in conjunction with a particular aspect, embodiment, or example of the invention are to be understood to be applicable to any other aspect, embodiment, or example of the invention. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

## Claims

1. A method of diagnosing an epithelial ovarian tumor with a poor prognosis in a subject, comprising:
contacting a sample containing ovarian tissue, ovarian cells or ovarian endothelial cells obtained from the subject with the epithelial ovarian tumor with a wild-type Zeste homologue 2 (EZH2) nucleic acid molecule or an anti-wild-type EZH2 antibody;
comparing expression of wild-type EZH2 detected in the sample obtained from the subject with the epithelial ovarian tumor to a control; and
detecting an at least 2-fold increase in expression of wild-type EZH2 relative to a control indicates the subject has a poor prognosis, thereby diagnosing the epithelial ovarian tumor in a subject with a poor prognosis.

2. The method of claim 1, wherein an at least 2-fold increase in expression of wild-type EZH2 as compared to the control indicates a stage III or stage IV epithelial ovarian tumor.

3. The method of claim 1, wherein an at least 2-fold increase in expression of wild-type EZH2 as compared to the control indicates a high grade epithelial ovarian tumor.

4. The method of claim 1, wherein expression of wild-type EZH2 is determined by polymerase chain reaction.

5. The method of claim 1, further comprising treating the subject with the epithelial ovarian tumor with a poor prognosis by administering to the subject an effective amount of a specific binding agent that preferentially binds to wild-type EZH2 and inhibits epithelial ovarian tumor growth in the subject, wherein the specific binding agent that preferentially binds to wild-type EZH2 is a wild-type EZH2 siRNA.

6. The method of claim 1, wherein the control is a sample obtained from a subject that has a non-metastatic ovarian tumor.

7. The method of claim 1, wherein the wild-type EZH2 nucleic acid molecule or an anti-wild-type EZH2 antibody is positioned on an addressable array.